Effect of Roughage-to-Concentrate Ratio and Lactic Acid Bacteria Additive on Quality, Aerobic Stability, and In Vitro Digestibility of Fermented Total Mixed Ration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Please received my comments, the aim of the work is novel and significant. Please consider reviewing the writing to improve understanding.

Thanks

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Needs revision to improve reading and understanding of the results. There are some sections more clearly stated as introduction and conlusion.

Thanks

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article "Effect of concentrate to forage ratio and lactic acid bacteria on the quality, aerobic stability, and in-vitro digestibility of fermented TMR" presents the results of laboratory experiment on the impact of different concentrate-to-forage ratios and the application of lactic acid bacteria on the quality, aerobic stability, and digestibility of a fermented total mixed ration based on oats and vetch. The authors conducted a 60-day fermentation of the mixture to analyze how varying ingredient proportions affect nutrient content, aerobic stability, and protein degradation indices. The research indicates that when the forage-to-concentrate ratio is 35:65, the nutritional quality and ruminal degradation rate of the fermented TMR are optimal. Although a higher level of concentrate feed can increase nutritional value, it may complicate the fermentation process and reduce aerobic stability; however, LAB additives can improve these aspects.

The study is interesting and has potentially significant practical value. Here are some suggestions that could enhance the quality of the manuscript:

Line 57: It would be beneficial to add detailed information about FTMR technology, explaining precisely what this technology entails, its advantages, and the specific issues that need to be addressed. This would help justify the necessity of the research conducted by the authors.

It is also recommended to include in to the Introduction section some characteristic of lactic acid bacteria in the context of feed fermentation and their role in the ensiling process of FTMR.

A clear research hypothesis is missing. It would be valuable for the authors to outline the assumptions they adopted when planning the experiment.

Material and methods section. Please provide a more detailed description of the silage production process so the reader has a fuller understanding of the experiment’s course.

Table 1. This table is unclear—if the feed components are presented on a dry matter basis (%), all components should sum to 100%. The table should be revised to clarify the proportion of each component in each experimental variant.

Line 3 in Table 1. Does it refer to “Oat-Pea” or “Oat-Vetch”? Please clarify this matter.

Line 204 - Please specify whether the concentrations of LA, AA, BA, and PA are expressed in % DM (dry matter) or FM (fresh matter)?

How was dry matter loss (DM loss) evaluated? Methodological details on this topic should be added.

Tables 4-11 - There are missing explanations for abbreviations below these tables, as well as information on interpreting the statistical analyses. Explanatory notes should be added to facilitate understanding.

Line 315 - It would be beneficial to explain why a pH higher than 4.2 is considered acceptable for fermented TMR.

Line 518 - The authors explain improved stability due to the addition of Lactobacillus plantarum and Lactobacillus pentosus, but they omit Lactobacillus buchnerii, a heterofermentative bacterial strain specifically used in inoculants to enhance aerobic stability in silages.

Information on the study's limitations is missing. The credibility of the work would be enhanced if the authors acknowledged potential limitations, such as the specificity of the raw materials used (e.g., oats and vetch), the scale of ensiling (laboratory scale), or storage duration (only 60 days).

 

The authors should discuss the practical application of their research findings in the context of animal husbandry and feed production optimization in more detail.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors

The study possesses scientific merit and is worthy of consideration for publication; however, several aspects require improvement to enhance its clarity and rigor. These points, outlined below, include suggestions to refine the methodology, presentation of results, and consistency in terminology. Addressing these aspects will strengthen the manuscript and provide a clearer understanding of the findings for the readers.

Title and Abstract

It is necessary to standardize the terminology and order of terms throughout the manuscript. In some sections, the ratio appears as "concentrate to forage," while in others it is presented as "forage to concentrate." For example, the title mentions "concentrate to forage," while in the main text, it appears as "forage to concentrate." It is recommended to choose one order and use it consistently. Additionally, considering that the authors used a mixture of forages and corn straw, the term "roughage" seems more appropriate than "forage." I suggest adopting the expression "roughage to concentrate ratio" throughout the manuscript to ensure terminological accuracy. Attention should be given to updating this in the text, tables, and figures (check carefully, as these terms appear throughout the manuscript).

In the abstract, it is recommended to specify the species of microorganisms used for the inoculation of total mixed rations (TMR), providing a clearer view of the biological additives employed. It would also be relevant to include some numerical values of the most important parameters in the abstract to highlight the main results obtained, thereby better showcasing the impact of the treatments and the significance of the findings to the reader.

Introduction

In the introduction, the authors should contextualize what is currently known about the use of inoculants in total mixed ration (TMR) silage production and identify the remaining knowledge gaps. Recent studies on TMR silages suggest that inoculants can significantly improve the chemical composition, but it is necessary to further explore how these benefits apply under different conditions. Therefore, the authors should include this information, discuss the possible changes in chemical fractions during fermentation, and highlight the anticipated benefits of the specific inoculants tested.

Additionally, it is essential to describe the function of each strain selected for the study, justifying their selection and detailing the expected effects of each strain on silage quality.

Another important point is that increasing the proportion of concentrate in TMR tends to raise the levels of dry matter (DM), soluble carbohydrates, and protein, while reducing fiber content, which directly impacts the quality of the fermentation process and the final nutritional value. These aspects need to be presented clearly, as they are essential for understanding the impact of TMR formulation on the final product.

Finally, it is recommended that the authors include, at the end of the introduction, the study hypothesis and a clear and direct objective, specifying what they intend to verify or demonstrate with the research. This will help guide the reader and establish the focus of the study

 

Material and Methods

 

Lines 91-96. The treatments need to be described more clearly, as the current wording is somewhat confusing. In fact, the authors conducted six treatments, considering three forage-to-concentrate ratios, each with and without the use of inoculants. Throughout the text, the authors refer to "treatments" only in terms of inoculant presence or absence, which is incorrect. This needs to be revised in the results tables, the statistical analysis, and the results description.

Additionally, it is important to specify the commercial brand of the inoculants if they were purchased from suppliers. If the inoculants were not commercially acquired, describe how they were obtained and stored to ensure the microbial populations cited in the text.

Correct "Bucherii" to "Buchneri."

Table 1. Standardize the order of the forage-to-concentrate ratio by listing forage first (e.g., 65:35 instead of 35:65). Replace "Refined material" to "Concentrate." Insert a line below the first "Total" and named it as "Concentrate," making it clear that the items listed below refer specifically to the composition of the concentrate.

If the same forage and concentrate were used for all treatments, there is no need to repeat the same information in separate columns for each treatment. Instead, include a footnote indicating that the same concentrate was used across all treatments. Replace "raw materials" with "TMR."

Use "Sodium bicarbonate" instead of "Baking soda."

Additionally, clarify the term “Gunk.” Is there a more commonly used or standardized name for this ingredient?

Provide detailed information on the mixing process of the ingredients for silage production. Specify whether the inoculants were diluted in water, the amount of water used, and the quantity of inoculant applied per bag. Describe how the inoculant was sprayed onto the forage.

The sentence “Triplicate samples of the untreated fresh forage were collected before ensiling” suggests that samples were collected only from the fresh forage. Were samples not also collected from the TMR silage? Please clarify.

Results and Discussion

 All abbreviations used in the tables should have their meanings provided in the footnotes of each table.

Tables 3 to 11 and Figures 1 and 2. The titles should be more descriptive, indicating that they refer to TMR produced with different forage-to-concentrate ratios, with and without inoculation.

Table 3. Why was there no significant difference between inoculants in the last row of the table, yet different letters appear in the columns for PA, AA, and LA?

The statement “LA content was significantly higher than that in the C3 group (P<0.05)”—is this correct? If so, please review the letters in the table to ensure they are consistent with the statistical results.

How were DM losses determined? This assessment is not described in the Materials and Methods section, and this information is essential for understanding the results.

Errors in the citation format were found in the text and need correction to ensure uniformity in referencing style.

The authors state that Clostridium bacteria were responsible for proteolysis and explain that the higher pH in C1 contributed to increased proteolysis. This is partially correct, as Clostridium growth is favored at higher pH levels. However, Clostridium does not tolerate low water activity, which aligns with the undetectable BA levels. In my view, filamentous fungi may have been the main contributors to this proteolysis. I suggest including references to support this interpretation and strengthen the discussion. 

Conclusion

"The forage-to-concentrate ratio is reversed (35:65)."

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

After reviewing the responses to my comments and the revised version of the manuscript, I am pleased to inform you that, in its current form, it is suitable for publication.

The revisions address the key points I previously raised, and the updated manuscript demonstrates clear improvements in structure, clarity, and scientific rigor. 

Congratulations on this accomplishment, and I look forward to seeing the final published version.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for the corrections made to the article. The changes significantly improved the clarity and quality of the work. In my view, the manuscript can now be accepted for publication.

Best regards,