Reducing Protein Content with and Without Yeast Probiotic Actisaf Sc 47 Supplementation in the Diet of Dairy Cow: Effects on Nitrogen Use, Digestibility, and Rumen Microbial Protein

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an interesting body of research. The experimental design needs revision regarding the statistical analysis presented in the manuscript. The materials and methods section lacks key details on measurement techniques, and the results section (e.g., milk yield and composition; RN) also omits essential information. Aside from issues with figure and table presentation, the interpretation of mean comparisons in the statistical analysis is highly confusing. Overall, it appears that key terminology is misused throughout the text and in the tables. The presentation of figures and tables may be inappropriate, and the items are so confusing that abbreviations and units are sometimes missing or used incorrectly. Additionally, tables do not clearly display the tests for comparing the treatment means.

Specific comments and suggestions for the sections Abstract, Introduction, Materials and Methods, and Results are in the comment box of the attached PDF file.

I am happy to review a re-written version of the manuscript if it has undergone an internal review by someone with a high standard of statistical analysis.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Absract

L 25 revise to a clear result e.g. L38, L39, L41  change "more significant" to "significantly" after revise statistical analysis model and the method of treatment mean comparison in the section Method=è We revised the abstract and did some precise modifications.

Introduction

L55-59 suggest consider to rewriting to more concise and clearlyè We modified the beginning of the introduction ( Line 58-59)

delete 14.8 appeared two timeè We did the modification

Materials and methods

3x3 Latin square design: L97 suggest to consult with statistician on the experimental design and ANOVA analysis should be "a replicated 3x3 Latin square design"è De discussed with a statistician. We did not replicate our Latin square design. We did one study with one Latin square design including 3 treatments with 2 cows in each treatment and period. è We added some explanations. Our statistical model include Treatment, periode, their interaction and cows

Milk yiled:  Suggest the author to add the milk yield and composition data report in the result sectionè The milk yiled performance was the subject of another publication which we wish to submit to the same journal. Since the milk yiled is automatically measured, we added it as information.

At the start of the trial: add animal information such as body weight, partity, HF%, sourceè We added some information in Animals, Experimental Design, and Diets Section

Table, Figure have to stand alone, suggest to add footnote what is abbreviate  "UFL, PDIE, PDIN" stand for.: UFL, PDIN, PDIEè We added information

L131-135 Suggest the author to add the milk yield and composition data report in the result section. Cows were milked twice a day at R2 07:00 h and 17:00 h and the total quantity of milk was automatically recorded daily. Throughout the measurement period (5 days), milk samples (100 ml) were collected from individual cow during morning and afternoon milking. The two samples were mixed and treated by preservative and stored at 4°C until analyzed for milk fat, true protein, lactose, and urea by infrared analysis (CIALSO, Auch, F-32000)è As milk quantity is automatically registered and as milk quality is routinely analyzed even even outside of testing, we added this part for more explanation on cow manipulation. The milk part including quantity, quality, fatty acid profile, feed and economic efficiency will be submitted as scientific paper in the same journal Animal open access

Statistical analysis

L 176 1) suggest to consult with statistician on the experimental design and ANOVA analysis should be "a replicated 3x3 Latin square design". This corrected change may influence to the power of statistical significants 2)add clearly of statistical ANOVA analysis model, 3) add the method of comparing the significant of treatment use in this study , consequently, Author had not clear the method of treatment means comparison & interpretation in the result sectionè our statistical design is simple and our study is not replicated. We used 3 treatments in 3 periods. We included in our statistical model all the factors that could impact on our variable responses. Also, we used previously published models using Latin square design. Nevertheless, we added more precision about the statistical software, the statistical model, the justification of sample size and data verification.

Results

Effects of Actisaf on nitrogen use

L 178-192 1)Author had no clear on the method of comparing the significant of treatment use in this study, suggest to add in the method section 2)Author had not clear how to do a statistical treatment means comparison & interpretation in the result section, suggest to revised all in the section 3.1 with consult to statisticianè We did modifications and added more precision and clarity in our analysis of results

Figure 2 1)Suggest to revise the presenting a separate all Figure and Table in the standard form as recommend by Animals author guideline (e.g. abbreviation list, footnote to standalone, all the results had describe detail in the method section) 2)describe RN estimation and add in the method sectionè We did the modifications and used a simple table.

L 199-211 1)Author had no clear on the method of comparing the significant of treatment use in this study, suggest to add in the method section 2)Author had not clear how to do a statistical treatment means comparison & interpretation in the result section, suggest to revised all in the section 3.2è We did the modifications and used a simple table

Discussion

L248 no data report on mlik productionè Data on milk performance will be submitted for publication in Animal Open access Journal

 

Conclusion

L 369-389 Conclusion, should agree with the result section, not replete result section e.g. L375-383è We did modification in conclusion

References

We verified and modified references

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

1. Revise sentence structure in line 62: “… [3] reported that Only 26 ± 38.2% …”. Correct citation placement and capitalization. Review similar citation formatting issues throughout the manuscript to ensure consistency with standard academic style.

2. A single-paragraph introduction reduces clarity. Divide the introduction into three main paragraphs: (1) problem formulation, (2) research gap supported by relevant citations, and (3) proposed solution, including hypothesis and research objectives. Revise accordingly to improve logical flow.

3. Clarify the expression of crude protein content: “… 14.5% CP%DM …” and “… with 14.5% CP%DM …”. Use a consistent and standard format, such as “14.5% CP (DM basis)”.

4. Table 1 appears truncated and requires correction. Ingredient composition expressed in kg/d is difficult to interpret directly. Convert values into percentage of dry matter (% DM) to improve readability and scientific clarity.

5. The supplementation level appears to be 5 g/d based on Table 1. Such a level is very low compared to the basal diet (e.g., corn, as-fed basis). Confirm whether Actisaf Sc 47 supplementation is indeed 5 g/d. Provide supporting references to justify the effectiveness of such dosage, especially in the absence of prior studies.

6. The experimental design using a 3×3 Latin Square Design (LSD) seems minimal. Confirm whether such replication meets recommended standards. Provide a valid reference to justify the use of this design.

7. Review phrasing: “… offered once a day (0900 h) …”. Ensure consistency with standard time notation (e.g., 09:00 h).

8. Evaluate whether mathematical expressions in Equations 1–7 require bold formatting. Confirm whether equations were generated using a proper equation editor in Word and ensure consistent formatting.

9. The statement “All analyses were performed using Minitab statistical software R version 19” is unclear. Verify whether Minitab, R, or both software packages were used. Revise the sentence to accurately reflect the analytical tools applied.

10. Figures 2 and 3 are presented as both bar charts and tables. Retain only one format to avoid redundancy. Include measures of variability (e.g., standard deviation) for each dataset. Ensure correct international decimal notation using a period (.) instead of a comma (,).

11. Conclude each paragraph in the Results section with a brief interpretation or plausible explanation of the findings, particularly for treatments involving LCP and LCP Actisaf.

12. Review the Discussion section for typographical errors and logical consistency. Ensure arguments remain coherent and scientifically supported.

13. Revise the Conclusion section. Avoid repeating general statements such as “Many studies …”. Focus directly on conclusions that address the research objectives.

Author Response

Comments and Suggestions for Authors

1. Revise sentence structure in line 62: “… [3] reported that Only 26 ± 38.2% …”. Correct citation placement and capitalization. Review similar citation formatting issues throughout the manuscript to ensure consistency with standard academic styleè OK. We did the modification in Lines 64-66
2. A single-paragraph introduction reduces clarity. Divide the introduction into three main paragraphs: (1) problem formulation, (2) research gap supported by relevant citations, and (3) proposed solution, including hypothesis and research objectives. Revise accordingly to improve logical flowè OK. We divided the introduction in 3 parts
3. Clarify the expression of crude protein content: “… 14.5% CP%DM …” and “… with 14.5% CP%DM …”. Use a consistent and standard format, such as “14.5% CP (DM basis)”è We used 14.5 CP% DM or 16.5 CP%DM in all the manuscript.
4. Table 1 appears truncated and requires correction. Ingredient composition expressed in kg/d is difficult to interpret directly. Convert values into percentage of dry matter (% DM) to improve readability and scientific clarityè We did the modification in Table 1. We converted kg/d to %DM
5. The supplementation level appears to be 5 g/d based on Table 1. Such a level is very low compared to the basal diet (e.g., corn, as-fed basis). Confirm whether Actisaf Sc 47 supplementation is indeed 5 g/d. Provide supporting references to justify the effectiveness of such dosage, especially in the absence of prior studiesè I agree with you as Live yeast is used also for starchy diet. But we used the registered dose of our product. Different study was conducted with this dose. Our registered dose has never been studied with diets lower in protein, that is why for this first trial, we decided to evaluate the effect if 5 g. Nevertheless, we can test other doses in the future.
6. The experimental design using a 3×3 Latin Square Design (LSD) seems minimal. Confirm whether such replication meets recommended standards. Provide a valid reference to justify the use of this design.==> We did this first study in our facilities, and we have a small herd composed of 7 cows. We know that using 6 cows in a parallel design makes no sens. That is why we used a Latin square design to improve the statistical power. Our study is a first step to validate or not the effect of our products before going to study with high number of animals.
7. Review phrasing: “… offered once a day (0900 h) …”. Ensure consistency with standard time notation (e.g., 09:00 h)è We did the modification and standardized the time
8. Evaluate whether mathematical expressions in Equations 1–7 require bold formatting. Confirm whether equations were generated using a proper equation editor in Word and ensure consistent formattingè The equations do not require bolding. So, we modified them. We used equation editor in Word to generate the equations
9. The statement “All analyses were performed using Minitab statistical software R version 19” is unclear. Verify whether Minitab, R, or both software packages were used. Revise the sentence to accurately reflect the analytical tools applied.==> We added more information and precision in the statistical analysis section.
10. Figures 2 and 3 are presented as both bar charts and tables. Retain only one format to avoid redundancy. Include measures of variability (e.g., standard deviation) for each dataset. Ensure correct international decimal notation using a period (.) instead of a comma (,)è We modified the figures to be more appropriate and understandable. We just used tables.
11. Conclude each paragraph in the Results section with a brief interpretation or plausible explanation of the findings, particularly for treatments involving LCP and LCP Actisafè We did modification by adding a brief conclusion
12. Review the Discussion section for typographical errors and logical consistency. Ensure arguments remain coherent and scientifically supportedè We verified the discussion and deleted non-useful information
13. Revise the Conclusion section. Avoid repeating general statements such as “Many studies …”. Focus directly on conclusions that address the research objectivesè We revised and modified the conclusion

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This study focuses on the effects of combining a low-protein diet with yeast-based probiotics in dairy cows. The research topic aligns with the industry’s demand for green and sustainable development in the dairy sector. Using six lactating Holstein cows as test subjects and employing a 3×3 Latin square experimental design, The results indicate that reducing the crude protein (CP) content in the dairy cow diet from 16.5% to 14.5% and supplementing with Actisaf Sc 47 can optimize nitrogen use efficiency, reduce nitrogen emissions, and improve fiber digestibility and ruminal microbial protein synthesis. This approach represents an effective strategy for achieving environmental sustainability in dairy farming; however, its impact on dairy cow production performance requires further validation through trials with larger sample sizes.

 

The specific suggestions for revision are as follows:

1、Line 24：The abbreviation “Sc47” is inconsistent with “Sc 47” used later in the text.

2、Line 29：Proposal to capitalize proper nouns: Latin square

3、Line 32：The 14.4% CP contradicts the 14.5% CP mentioned later; this needs to be consistent.

4、Line59：Punctuation errors. economic gain, [1].

5、Line62：The citation format is incorrect.   [2]. [3]

6、Line70/272：Chemical formula format error，^-，₃

7、Line76：The writing is repeated.16.7 to 14.8 14.8%

8、Line96-113：Experimental animal information is only marked “92.5 ± 2.59 days in milk and 38.71 ± 5.92 kg of milk”，The basic information such as parity, body weight and body condition score ( BCS ) of dairy cows were not explained, and the health status of dairy cows during the test was not clarified, which needed to be supplemented to ensure the repeatability of the test.

9、Line103：The value is inconsistent with the following and summary. 16.6% of crude protein

10、Line104/159/307：Format error.  14.5% CP%DM /BW 0.75 /Lima al, 2023

11、Line118-126：Diet feeding amount only shows “fed once a day”，The specific feeding amount ( such as the percentage of body weight ) and the control range of residual feed rate were not marked, and the drinking water management mode of dairy cows during the test was not explained.

12、Line120：The first letter capital of the chart title.table 1

13、Line122：clerical error. Crude fiber (%DM°)

14、Line122：For LCPA in Table 2, the full text needs to be unified as LCPActisaf.

15、Line126/143：Time format error.0900h

16、Line141/160：Urine samples were collected by ' point sampling method ', but the time and method of body weight ( BW ) measurement were not described. BW is a key index to calculate urine output and purine derivative absorption, which needs to be supplemented. Fecal sample collection only marked ' 500g / time ', did not explain the number of daily sampling, and did not mention the homogeneous treatment method after fecal sample mixing, which needs to be improved.

17、Line163：Check the unit for errors. 150 mg

18、Line167/197、215/201/234、243/236/246/287：spelling mistake. aide insoluble ash /treatement effect/digesibility/environemental/address/investigare/Northen

19、Line189-192：“Retained nitrogen（RN）” It is mentioned for the first time, but the calculation method is not clear in the text and needs to be supplemented.

20、Line192-197：The SEM value of nitrogen utilization related indicators in figure 2 is larger, and the reasons for this phenomenon need to be analyzed in the result part ( such as the small number of experimental animals and large individual differences ).

21、Line212：The data comma in the table is changed to decimal point.
22、Line266/277,303：Misuse of prepositions. from 16.5% et 14.5%  /on the rumen

23、Line270/295：Fixed collocation errors. in a combination

24、Line386-387：The conclusion here refers to “the use of Actisaf Sc 47 may have a positive impact on the production performance of dairy cows ”, but this study did not determine the production performance indicators such as milk yield, milk composition ( milk protein, milk fat ), etc. The speculative expression needs to be deleted or the experimental results supplemented.

Author Response

Comments and Suggestions for Authors

This study focuses on the effects of combining a low-protein diet with yeast-based probiotics in dairy cows. The research topic aligns with the industry’s demand for green and sustainable development in the dairy sector. Using six lactating Holstein cows as test subjects and employing a 3×3 Latin square experimental design, The results indicate that reducing the crude protein (CP) content in the dairy cow diet from 16.5% to 14.5% and supplementing with Actisaf Sc 47 can optimize nitrogen use efficiency, reduce nitrogen emissions, and improve fiber digestibility and ruminal microbial protein synthesis. This approach represents an effective strategy for achieving environmental sustainability in dairy farming; however, its impact on dairy cow production performance requires further validation through trials with larger sample sizes.

 

The specific suggestions for revision are as follows:

1、Line 24：The abbreviation “Sc47” is inconsistent with “Sc 47” used later in the textè We did the modification in all the manuscript (Actisaf Sc 47)

2、Line 29：Proposal to capitalize proper nouns: Latin squareè We did the modification (Lines 32 and 109)

3、Line 32：The 14.4% CP contradicts the 14.5% CP mentioned later; this needs to be consistentè we did the modifications in all the manuscript.

4、Line59：Punctuation errors. economic gain, [1]è We modified the sentence.

5、Line62：The citation format is incorrect.   [2]. [3]è We did modifications in lines 63 and 66.

6、Line70/272：Chemical formula format error，^-，₃                 è we did modification in Line 72

7、Line76：The writing is repeated.16.7 to 14.8 14.8%==> We corrected in line 78

8、Line96-113：Experimental animal information is only marked “92.5 ± 2.59 days in milk and 38.71 ± 5.92 kg of milk”，The basic information such as parity, body weight and body condition score ( BCS ) of dairy cows were not explained, and the health status of dairy cows during the test was not clarified, which needed to be supplemented to ensure the repeatability of the testè We added more information in lines 100 to 131.

9、Line103：The value is inconsistent with the following and summary. 16.6% of crude proteinè We corrected in line 117

10、Line104/159/307：Format error.  14.5% CP%DM /BW 0.75 /Lima al, 2023è We did modifications in line 115, 116 and all the manuscript. We changed BW075 by BW^0.75 and we deleted Lima et al

11、Line118-126：Diet feeding amount only shows “fed once a day”，The specific feeding amount (such as the percentage of body weight) and the control range of residual feed rate were not marked, and the drinking water management mode of dairy cows during the test was not explainedè We added information in lines 146,148

12、Line120：The first letter capital of the chart title.table 1è We modified in line 144 Table 1

13、Line122：clerical error. Crude fiber (%DM°)è we corrected in Table 1

14、Line122：For LCPA in Table 2, the full text needs to be unified as LCPActisafè We did modification in table 2

15、Line126/143：Time format error.0900hè we did modification in 160,167, 181 to 184.

16、Line141/160：Urine samples were collected by ' point sampling method ', but the time and method of body weight ( BW ) measurement were not described. BW is a key index to calculate urine output and purine derivative absorption, which needs to be supplemented. Fecal sample collection only marked ' 500g / time ', did not explain the number of daily sampling, and did not mention the homogeneous treatment method after fecal sample mixing, which needs to be improvedè we added more information in Lines 202 to 213. Cows were weighed at the beginning of each period.

17、Line163：Check the unit for errors. 150 mgè We did the modification in line 208

18、Line167/197、215/201/234、243/236/246/287：spelling mistake. aide insoluble ash /treatement effect/digesibility/environemental/address/investigare/Northenè we corrected the spelling mistake in all the manuscript

19、Line189-192：“Retained nitrogen（RN）” It is mentioned for the first time, but the calculation method is not clear in the text and needs to be supplementedè We added formula footnot table 2

20、Line192-197：The SEM value of nitrogen utilization related indicators in figure 2 is larger, and the reasons for this phenomenon need to be analyzed in the result part ( such as the small number of experimental animals and large individual differences )è We added information in Lines 259-262

21、Line212：The data comma in the table is changed to decimal pointè We did correction in all tables
22、Line266/277,303：Misuse of prepositions. from 16.5% et 14.5%  /on the rumenè We did modification in line 334

23、Line270/295：Fixed collocation errors. in a combinationè We did modification in all the manuscript

24、Line386-387：The conclusion here refers to “the use of Actisaf Sc 47 may have a positive impact on the production performance of dairy cows ”, but this study did not determine the production performance indicators such as milk yield, milk composition ( milk protein, milk fat ), etc. The speculative expression needs to be deleted or the experimental results supplementedè We deleted information regarding production performance. This part is ready to be submitted as another scientific paper in the same journal Animal open access.

 

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

Comments to the authors of manuscript animals-4222071 entitled "Reducing protein content with and without yeast probiotic Actisaf Sc 47 supplementation in the diet of dairy cow: effect on nitrogen use, digestibility and rumen microbial protein."

Manuscript addresses a relevant nutritional and environmental question in dairy cows, but the current version suffers from major methodological and interpretative limitations. The experimental design does not allow isolation of the specific effect of Actisaf Sc 47 because no 16.5% CP + Actisaf group was included. The description of the Latin square is incomplete, the sample size is very small, statistical reporting is insufficient, ARRIVE reporting is inadequate, and several conclusions overstate the data, especially regarding rumen microbial protein synthesis and performance. Substantial revision is required before the manuscript can be considered further.

1. Missing 16.5% CP + Actisaf group
2. Incomplete and unclear description of the Latin square design (6 cows, no clarification of two squares)
3. Very small sample size (n = 6)
4. No sample size justification (no power analysis)
5. No clearly defined primary outcome
6. Insufficient description of randomization (no sequence allocation details)
7. No information on blinding
8. Missing inclusion/exclusion criteria
9. Inadequate compliance with ARRIVE guidelines
10. Change in housing conditions (free-stall → tie-stall) without control of its effect
11. Unclear period structure (adaptation / transition phases)
12. Incomplete description of the statistical model for a Latin square design
13. No inclusion of square effect in the statistical model
14. Unclear experimental unit
15. No clarification of repeated measures handling (daily data vs averaged data)
16. No reporting of model assumption checks (normality, homogeneity)
17. No description of post hoc testing procedures
18. Overuse of “trends” (p > 0.05)
19. Overinterpretation of non-significant results
20. Inconsistency between results and conclusions (RMP, performance)
21. Lack of full production data despite conclusions on performance
22. Overinterpretation of Actisaf effects without appropriate design
23. Mechanistic speculation without supporting experimental data
24. Inconsistent group nomenclature (LCPActisaf / LCPA)
25. Errors and lack of clarity in tables (Table 1, units, composition)
26. Poor data presentation (figures used as tables)
27. Lack of individual data presentation with n = 6
28. Errors in statistical software description
29. Language issues (grammar, syntax, spelling)
30. Inconsistent and incorrect reference formatting
31. Inappropriate conflict of interest declaration
32. Lack of transparency regarding funding and author affiliations
33. Inconsistencies between abstract, results, and conclusions
34. No clear separation of CP vs Actisaf effects
35. Overstated conclusions in Discussion and Conclusion sections

Author Response

Comments and Suggestions for Authors

Comments to the authors of manuscript animals-4222071 entitled "Reducing protein content with and without yeast probiotic Actisaf Sc 47 supplementation in the diet of dairy cow: effect on nitrogen use, digestibility and rumen microbial protein."

Manuscript addresses a relevant nutritional and environmental question in dairy cows, but the current version suffers from major methodological and interpretative limitations. The experimental design does not allow isolation of the specific effect of Actisaf Sc 47 because no 16.5% CP + Actisaf group was included. The description of the Latin square is incomplete, the sample size is very small, statistical reporting is insufficient, ARRIVE reporting is inadequate, and several conclusions overstate the data, especially regarding rumen microbial protein synthesis and performance. Substantial revision is required before the manuscript can be considered further.

1. Missing 16.5% CP + Actisaf groupè Very interesting remark. We thought about it before starting the trial. We don’t have enough cows (7 cows in our facility) to add this interesting group. That is why we chose to use 14.5%CP alone to compare it with 16.5% CP and 14.5%CP with Actisaf to evaluate the potential effects of Actisaf. That was the only one option to validate our hypothesis. For the future, if we repeat the trial externally, we will compare 4 groups.
2. Incomplete and unclear description of the Latin square design (6 cows, no clarification of two squares)è We used 3 x 3 Latin square design with 3 treatments and 3 periods. In each treatment we used 2 cows. We did modification and precise description in Material and methods part
3. Very small sample size (n = 6)è We are truly aware of the limit of the sample size. We are completely convinced of the importance of the sample size for robustness and relevance of data. The issue is that we don’t have enough cows in our facility. That is why we used 6 cows. Also, it is an exploratory trial before going to trials with high number of animals with external research center or commercial farms.
4. No sample size justification (no power analysis)è we added information in the manuscript (See statistical analysis section)
5. No clearly defined primary outcomeè We added more information about outcomes to the summary section
6. Insufficient description of randomization (no sequence allocation details)è we added more information in Materials and Methods/ Animals, Experimental Design, and Diets section
7. No information on blindingè I’m not sure that we can add the following information in the manuscript. As a ruminant scientific officer, I have drafted the protocol and validated it with scientific colleagues and laboratories and animal technicians. All sampling procedures were done by dedicated people. The protocol was validated internally by Phileo r&d team. The allocation of animals between groups was done by me as a scientist with respect of allocation criteria (Milk yield, day in milk and parity). Formulation of rations was done by an external expert. The management of animals during the trial (feeding, sampling) was done by an R&D animal and laboratory technician. All the people that followed the trial have the certificate of authorization for animal experimentation. Data analysis was done by me with the support of our statistician. I used the same statistical model from previous studies using Latin square design.

 

8. Missing inclusion/exclusion criteriaè We added some information in Materials and Methods/ Animals, Experimental Design, and Diets section
9. Inadequate compliance with ARRIVE guidelinesè We added information in Materials and Methods/ Animals, Experimental Design, and Diets section

1. Change in housing conditions (free-stall → tie-stall) without control of its effectè Cows are well adapted to the two housing conditions because they were milked twice a day. Two days before the start of the 5-day sampling period, the cows are tied up to allow them to adapt to being tied up. They are monitored (feed intake, behavior, milk production) during these two days.

 

11. Unclear period structure (adaptation / transition phases)è information is given in figure 1. We added some information about transition phases in in Materials and Methods/ Animals, Experimental Design, and Diets section

 

1. Incomplete description of the statistical model for a Latin square designè We added more precision regarding statistical model.
2. No inclusion of square effect in the statistical modelè In the statistical model, we used all the parameters that can influence the parameters to be explained. We also followed some previous studies that used Latin square design. The effect of squares may be explained by the period and cow.
3. Unclear experimental unitè the experimental unit is the animal (individual cow).
4. No clarification of repeated measures handling (daily data vs averaged data)è We really understand this point. Our data were averaged. For example, we collected 6 urine and fecal samples at different time points and it was possible to analyse them individually and then run a statistical model with repeated measures. We followed the recommendations of previous studies which combine the different samples to obtain a single representative sample.
5. No reporting of model assumption checks (normality, homogeneity)è We added information in statistic section
6. No description of post hoc testing proceduresè We added information in statistic section
7. Overuse of “trends” (p > 0.05)è Trends for difference is defined when p value is between 0.05 and 0.1.
8. Overinterpretation of non-significant resultsè We did modification with more precision in abstract and results section
9. Inconsistency between results and conclusions (RMP, performance)è We modified the conclusion
10. Lack of full production data despite conclusions on performanceè data on performance will be submitted for publication in the same journal. Animal open access. We deleted information about performance from the conclusion
11. Overinterpretation of Actisaf effects without appropriate designè We know the biological limit of such design. But animals were used under same conditions and the main factor of variation of response variables is the diet with and without Actisaf. The importance of conducting trials with more animals to confirm the positive effects of Actisaf was highlighted.
12. Mechanistic speculation without supporting experimental dataè I don’t understand this remark.
13. Inconsistent group nomenclature (LCPActisaf / LCPA)è We corrected in all the manuscript
14. Errors and lack of clarity in tables (Table 1, units, composition)è we did modification in table 1
15. Poor data presentation (figures used as tables)è We did modification and used understandable tables
16. Lack of individual data presentation with n = 6è The data presented in tables are for N=6
17. Errors in statistical software descriptionè We added more information about the statistical analysis
18. Language issues (grammar, syntax, spelling)è We did our best to correct tha grammar and syntax….
19. Inconsistent and incorrect reference formattingè We verified all the references and modified those who are not well formatted
20. Inappropriate conflict of interest declarationè There is no conflict of interest regarding our study. We used the same declaration in our own previous publications and also other publications
21. Lack of transparency regarding funding and author affiliationsè The study was defined and followed by scientist from Phileo with high expertise in the field. The trial is done in Phileo’s experimental unit. I don’t understand the point of lack of transparency. All of authors are from Phileo except Axel Blancou who work for Phileo as replacement service
22. Inconsistencies between abstract, results, and conclusionsè We modified Abstract, results and conclusion to be consistent.
23. No clear separation of CP vs Actisaf effectsè We added more precision
24. Overstated conclusions in Discussion and Conclusion sectionsè We did modifications in all the manuscript

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author requires a major version. The materials and methods are limited to the experimental design and data collection; the resulting limited data are not fully reported in this article (e.g., milk yield and composition), omitting essential information. The presentation of the result tables may be inappropriate. My detailed suggestions are as follows.

L248 suggests that the author report data on milk yield and composition in this article to be a complete research work because this 4 Tables results are weak and do not report all the data, which may raise research ethics issues.

L259-263 move to discussion section

L264 add all abbreviations in the Table to footnote //Suggest to revise the presentation of a separate all Figure and Table in the standard form as recommended by Animals author guideline (e.g. abbreviation list, footnote to standalone, all the results had described detail in the method section)//

L265-266, and all Table footnotes, correct "column" to "row" in all tables.

L283-285 move to the discussion section

L286 Suggest to revise the presentation of a separate Figure and Table in the standard form as recommended by Animals author guideline (e.g. abbreviation list, footnote to standalone, all the results had described detail in the method section)

L288-289, and all Table footnotes, correct "column" to "row" in all tables.

L298 Suggest to revise the presentation of a separate Figure and Table in the standard form as recommended by Animals author guideline (e.g., abbreviation list, footnotes to stand alone, all the results had described detail in the method section)

L304 Suggest that the author discuss the limitations of this study method, urine spot sampling compared to total collection methods, the gold standard, and the variation that could lead to type I error in statistical analysis

Author Response

The author requires a major version. The materials and methods are limited to the experimental design and data collection; the resulting limited data are not fully reported in this article (e.g., milk yield and composition), omitting essential information. The presentation of the result tables may be inappropriate. My detailed suggestions are as followsè We mentioned that cows are automatically milked and data on milk performance will be published in a second paper. We deleted information about milk.

- The English has been revised and corrected by MDPI English Editing of Animal Journal

L248 suggests that the author report data on milk yield and composition in this article to be a complete research work because this 4 Tables results are weak and do not report all the data, which may raise research ethics issuesè We really understand this remark. But we have a lot of data on Milk yield, efficiency, milk fatty acid profiles and economic efficiency we want tou submit it as a separate publication. We deleted information on milk in this paper

L259-263 move to discussion sectionè OK. We moved this part to discussion sectionè Line 366-370

L264 add all abbreviations in the Table to footnote //Suggest to revise the presentation of a separate all Figure and Table in the standard form as recommended by Animals author guideline (e.g. abbreviation list, footnote to standalone, all the results had described detail in the method section)//è We made all the modifications in all tables. Tables were also The tables and figures have been formatted by MDPI English editing of Animal journal

L265-266, and all Table footnotes, correct "column" to "row" in all tablesè OKè We made modifications in all tables and figures

L283-285 move to the discussion sectionè We made modification in discussion section

L286 Suggest to revise the presentation of a separate Figure and Table in the standard form as recommended by Animals author guideline (e.g. abbreviation list, footnote to standalone, all the results had described detail in the method section)è We followed Animal open access guidelines and all the tables and figures were formatted by MDPI English editing of Animal journal

L288-289, and all Table footnotes, correct "column" to "row" in all tablesè OKè We made modifications in all tables and figures

L298 Suggest to revise the presentation of a separate Figure and Table in the standard form as recommended by Animals author guideline (e.g., abbreviation list, footnotes to stand alone, all the results had described detail in the method section)è We followed Animal open access guidelines and all the tables and figures were formatted by MDPI English editing of Animal journal

L304 Suggest that the author discuss the limitations of this study method, urine spot sampling compared to total collection methods, the gold standard, and the variation that could lead to type I error in statistical analysisè We discussed briefly this point. Lines 401-405

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The author has made revisions properly and precisely based on my feedback. I want to remind you again to re-check the author's name and other details to confirm they are correct. After that, proofread everything once more to ensure accuracy, paying special attention to typography and any logical inconsistencies in the writing.

Author Response

- The English has been revised and corrected by MDPI English Editing of Animal Journal

1. Revise sentence structure in line 62: “… [3] reported that Only 26 ± 38.2% …”. Correct citation placement and capitalization. Review similar citation formatting issues throughout the manuscript to ensure consistency with standard academic styleè OK. We made this modification in Lines 66
2. A single-paragraph introduction reduces clarity. Divide the introduction into three main paragraphs: (1) problem formulation, (2) research gap supported by relevant citations, and (3) proposed solution, including hypothesis and research objectives. Revise accordingly to improve logical flowè OK. We divided the Introduction into three parts.
3. Clarify the expression of crude protein content: “… 14.5% CP%DM …” and “… with 14.5% CP%DM …”. Use a consistent and standard format, such as “14.5% CP (DM basis)”è We used 14.5 CP% DM or 16.5 CP%DM throughout the manuscript.
4. Table 1 appears truncated and requires correction. Ingredient composition expressed in kg/d is difficult to interpret directly. Convert values into percentage of dry matter (% DM) to improve readability and scientific clarityè We made this modification to Table 1. We converted kg/d to %DM.
5. The supplementation level appears to be 5 g/d based on Table 1. Such a level is very low compared to the basal diet (e.g., corn, as-fed basis). Confirm whether Actisaf Sc 47 supplementation is indeed 5 g/d. Provide supporting references to justify the effectiveness of such dosage, especially in the absence of prior studiesè I agree with you as live yeast is also used for starchy diets. However, we used the registered dose of our product. A different study was conducted with this dose. Our registered dose has never been studied with diets lower in protein; that is why for this first trial, we decided to evaluate the effect of 5 g. Nevertheless, we can test other doses in the future.
6. The experimental design using a 3×3 Latin Square Design (LSD) seems minimal. Confirm whether such replication meets recommended standards. Provide a valid reference to justify the use of this design.==> We did this first study in our facilities, and we have a small herd composed of seven cows. We know that using six cows in a parallel design makes no sense. That is why we used a Latin square design to improve the statistical power. Our study is a first step in validating the effect of our products before studying a higher number of animals.
7. Review phrasing: “… offered once a day (0900 h) …”. Ensure consistency with standard time notation (e.g., 09:00 h)è We made this modification and standardized the time.
8. Evaluate whether mathematical expressions in Equations 1–7 require bold formatting. Confirm whether equations were generated using a proper equation editor in Word and ensure consistent formattingè The equations do not require bolding. So, we modified them. We used the equation editor in Word to generate the equations.
9. The statement “All analyses were performed using Minitab statistical software R version 19” is unclear. Verify whether Minitab, R, or both software packages were used. Revise the sentence to accurately reflect the analytical tools applied.==> We added more precise information to the Statistical Analysis section.
10. Figures 2 and 3 are presented as both bar charts and tables. Retain only one format to avoid redundancy. Include measures of variability (e.g., standard deviation) for each dataset. Ensure correct international decimal notation using a period (.) instead of a comma (,)è We modified the figures to be more appropriate and understandable. We just used tables.
11. Conclude each paragraph in the Results section with a brief interpretation or plausible explanation of the findings, particularly for treatments involving LCP and LCP Actisafè We made this modification by adding a brief conclusion.
12. Review the Discussion section for typographical errors and logical consistency. Ensure arguments remain coherent and scientifically supportedè We verified the discussion and deleted non-useful information.
13. Revise the Conclusion section. Avoid repeating general statements such as “Many studies …”. Focus directly on conclusions that address the research objectivesè We revised and modified the Conclusions.