Effects of Rumen-Degradable Starch Levels on In Vitro Rumen Fermentation and Microbial Protein Synthesis in Alfalfa Silage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I find the manuscript very interesting, it makes important contributions, it shows how starch degradation takes place over time in the rumen of animals, however, in order to be published, some remarks should be made in the paper.

Comments for author File: Comments.pdf

Author Response

General Comments to Author:

Co1: I find the manuscript very interesting, it makes important contributions, it shows how starch degradation takes place over time in the rumen of animals, however, in order to be published, some remarks should be made in the paper.

Re: I am deeply grateful for your acknowledgment of our research and your valuable feedback. These comments are all valuable for the revising and improving of this paper, and also have important guiding significance to our research. We have studied comments carefully and made the revision which we hope meet with the reviewers’ requirements.

Co2: Experimental design in abstract

Re: Thank you very much for pointing out this important issue. We have carefully modified the abstract experimental design section (Line 18-26).  

Co3: It is necessary to mention sown area, sowing density, type of fertilisation, how many days after sowing the alfalfa was cut.

Re: Thank you for your valuable comment. We have added a description in the materials (Line 91-95).

Co4: How the inclusion of the different levels of ingredients was determined?

Re: We first determined the nutritional levels of raw materials, and then formulated diets with isoenergetic isonitrogen. After preparation, the nutrient levels of different diets were determined again. The nutrient composition of the substrate raw materials is shown in Table 1 (Line 113).

Co5: The meaning of the abbreviations is missing as a footnote to the table: DM, DE, CP, ADF, NDF, EE, RDS, RDP.

Re: Thank you very much for pointing out this important issue. We have added the abbreviations in the footnote to the table (Line 122-124).

Co6: What was the mathematical model used.

Re: Thank you for your detailed review and valuable suggestions on this article. We have added a description to the materials. We first processed one-way ANOVA on our date, and then by Duncan's multiple range test evaluate the differences among treatments. Meanwhile, regression (REG) analysis was conducted to evaluate the linear and quadratic effects of the increasing levels of RDS on the fermentation parameters (Line 175-181).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Find the paper in the attached file

Comments for author File: Comments.pdf

Author Response

Co1: The paper aimed to evaluate the dynamic changes of different rumen degradable starch levels on in vitro fermentation of alfalfa silage.

The paper is well written; the methodologies are adequate, and the results are well depicted and discussed with consistent conclusions.

THE VERSION OF PAPER I RECEIVED LACKS IN LINE NUMBERS; THEREFORE I WILL CITE SOME SENTENCE OF THE PAPER TO INDICATE WHERE EVENTUALLY CORRECTIONS AND/OR ADDITIONS ARE REQUIRED

Re: I am deeply grateful for your acknowledgment of our research and your valuable feedback. These comments are all valuable for the revising and improving of this paper, and also have important guiding significance to our research. We have added line numbers to the full text and made the revision which we hope meet with the reviewers’ requirements.

Co2: Material and Methods

Please report in Table 1 the symbol of percentage as well as the unit of measure for DE.

Re: Thank you very much for pointing out this important issue. We have added the unit of measure for DE in Table 1 (Line 117).

Co3: In vitro experiment: please give diet information of donor sheep (at least crude protein and NDF percentage).

Re: We appreciate your insight and added the nutrient levels of donor sheep (Line 129).

Co3: Discussion

After the sentence “Xu et al. [20] observed that as the ratio of wheat in the substrate increased, the rate and extent of gas production also increased linearly in vitro” please add as follows:

“Accordingly, Calabro et al. (2005a) comparing fresh and dried silage found that higher availability of energy and N improves the microbial growth mainly at the beginning of incubation resulting in more rapid fermentation and increased gas production”.

After the sentence ”However, despite limited differences in the Acetate/Propionate ratio between RDS and alfalfa silage in the present study, TVFA in incubation medium increased with increasing RDS level in the substrate, indicating that alfalfa silage provided a good nitrogen source for microorganisms, produced more amylase, therefor increased the yield of TVFA” please add as follows:

“In addition, even if the release of CO2 from microbial metabolism is associate with acetate production (Calabrò et al., 2005b), the slight difference in the Acetate/Propionate ratio support the significant increase of gas production with the level of RDS in the substrate”.

Calabrò, S.; Cutrignelli, M.I.; Bovera, F.; Piccolo, G.; Infascelli, F. In vitro fermentation kinetics of carbohydrate fractions of fresh forage, silage and hay of Avena sativa. J. Sci. Food Agric. 2005b, 85, 1838–1844.

Re: Thank you for your valuable comment. We are also aware that there is still missing for interpretation of the results. Therefore, we have added the above description to the discussion (Line 244-247, 297-299).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Authors should use the template provided by the journal to prepare their paper.

How did the authors arrive at the levels of rumen degradable starch tested in this study?

Describe this in the methodology

Replace volatile fatty acids with short-chain fatty acids throughout the paper

Remove the words that are present in the paper title from the keywords.

A hypothesis should be included in the introduction

At the beginning of the material and methods, a topic must be included that addresses where the experiment was conducted, as well as where the laboratory analyses were performed, describing the location (latitude, longitude, altitude, precipitation, etc.)

What was the alfalfa harvest area like? How was the harvest performed? At what height in relation to the ground?

DM (105 °C), crude protein (CP), ether extract (EE), ash, acid detergent fiber (ADF), and neutral detergent fiber (NDF) - describe the methods used in more detail

A table with the composition of the ingredients used should be inserted before Table 1. Composition and nutritional level of substrate in vitro experiment (air-dry basis)

Table 1 - all acronyms should be described in the caption Check this in all tables

Table 1 - How was the DE obtained? This is not included in the methodology

Table 1 - Insert the energy levels of the treatments

In the in vitro analysis, how many blanks were used? Why did the authors use only 3, 6, 12, and 24 h of incubation? What methodology was adopted to define the incubation times?

All methods should be better detailed so that the experiment can be replicated. Simply mentioning what was done is very vague and this has already been done in the paper's abstract.

Make, model, city and country of manufacture must be entered for all equipment used

Data processing should be detailed in more detail, how were the data processed? Were they normalized? How were repeated measurements analyzed over time? In relation to regression analysis? How was significance chosen? Based on the correlation coefficient? .... The statistical model must be entered

 

 

 

Author Response

Co1: Authors should use the template provided by the journal to prepare their paper.

Re: We appreciate your insight and used the templates to prepare the paper.

Co2: How did the authors arrive at the levels of rumen degradable starch tested in this study?

Describe this in the methodology

Re: Thank you for your valuable comment. Rumen degradable starch levels were calculated based on raw materials and starch content. Formulas and references were listed in the method (Line 159-165)..

Co3: Replace volatile fatty acids with short-chain fatty acids throughout the paper

Re: Thank you for your valuable comment. We have replaced volatile fatty acids with short-chain fatty acids throughout the paper.

Co4: Remove the words that are present in the paper title from the keywords.

Re: Thank you for your valuable comment. We revised the keywords.

Co5: A hypothesis should be included in the introduction

Re: Thank you for your valuable comment. We have added a hypothesis in the Introduction (Line 78-80).

Co6: At the beginning of the material and methods, a topic must be included that addresses where the experiment was conducted, as well as where the laboratory analyses were performed, describing the location (latitude, longitude, altitude, precipitation, etc.)

Re: Thank you for your valuable comment. We have added this description in the materials section (Line 85-90).

Co7: What was the alfalfa harvest area like? How was the harvest performed? At what height in relation to the ground?

Re: Thank you for your valuable comment. We have added this description in the materials section (Line 91-95).

Co8: DM (105 °C), crude protein (CP), ether extract (EE), ash, acid detergent fiber (ADF), and neutral detergent fiber (NDF) - describe the methods used in more detail

Re: Thank you for your valuable comment. We have added this description in the materials section (Line 99-107).

Co9: A table with the composition of the ingredients used should be inserted before Table 1. Composition and nutritional level of substrate in vitro experiment (air-dry basis)

Re: Thank you for your valuable comment. We have inserted a table of raw materials before Table 1 in the materials section (Line 113).

Co10: Table 1 - all acronyms should be described in the caption Check this in all tables

Re: Thank you for your valuable comment. We have added this description in the all tables (Line 88-90).

Co11: Table 1 - How was the DE obtained? This is not included in the methodology

Re: Thank you for your valuable comment. The digestible energy (DE) was calculated by referring to the raw materials [23] (Line 107).

[23] Sauvant, D.; Perez, J.M.; Tran, G. Tables of Composition and Nutritional Value of Feed Materials: Pig, Poultry, Sheep, Goats, Rabbits, Horses, Fish. Agricultural and Food Sciences. 2004, 10.

Co12: Table 1 - Insert the energy levels of the treatments

Re: Thank you for your valuable comment. We have inserted the energy levels of the treatments in Table 1 and 2 (Line 88-90).

Co13: In the in vitro analysis, how many blanks were used? Why did the authors use only 3, 6, 12, and 24 h of incubation? What methodology was adopted to define the incubation times?

Re:  Thank you very much for pointing out this important issue. We are also aware of the oversight in not including a blank. We understand your point that it could further strengthen our findings. However, we would like to emphasize that even despite the absence of a blank, our manuscript provides a validity of our experimental results. Similarly, most in vitro experiments we referenced also lacked a blank [1-3]. In addition, the incubation times selected for our in vitro experiments—3, 6, 12, and 24 hours—were set in geometric progression. The growth and activity of rumen microorganisms was the highest after feeding for 3h [4], followed by doubling the incubation time until fermentation was stopped at 24 hours [1-3]. Before initiating our experiment, we reviewed previous in vitro studies and found that most of them fermented for 24 hours, with a decrease in fermentation rate and less gas and acid production after 48 hours of fermentation [5]. Unlike in vivo substrate degradation, due to the absence of dynamic nutrient influx and efflux, longer fermentation times may lead to incorrect conclusions. The optimal time to terminate in vitro fermentations is after 18–24 hours of incubation [5]. Therefore, we selected 3, 6, 12, and 24 hours as the incubation time points.

[1] Zhao, W.; Abdelsatta, M.M.; Wang, Xin.; Zhang, N.; Chai, J. In Vitro Modulation of Rumen Fermentation by Microbiota from the Recombination of Rumen Fluid and Solid Phases. Microbiology Spectrum. 2022, 11, 1.

[2] Xue, C.; Wang, Y.; He, Z. et al. Melatonin disturbed rumen microflora structure and metabolic pathways in vitro.Microbiology Spectrum, 2023, 11(6).

[3] An, J.; Shen, W.; Liu, H.; Yang, C.; Chen, K.; Yuan, Q.; et al. Comparison of the effects of rumen-protected and unprotected l-leucine on fermentation parameters, bacterial composition, and amino acids metabolism in in vitro rumen batch cultures. Frontiers in Microbiology. 2023.

[4] Belanche, A.; Palma-hidalgo, J.M.; Nejiam, et al. In vitro assessment of the factors that determine the activity of the rumen microbiota for further applications as inoculum. Journal of the Science of Food and Agriculture.2019, 99(1):163-172.

[5] Dhakal, Rajan.; Neves, A.; Sapkota, Rumakanta.; Khanal, Prabhat.; Ellegaard-Jensen, Lea.; Winding, Anne.; Hansen, H. Temporal dynamics of volatile fatty acids profile, methane production, and prokaryotic community in an in vitro rumen fermentation system fed with maize silage. Frontiers in Microbiology. 2024, 15.

Co14: All methods should be better detailed so that the experiment can be replicated. Simply mentioning what was done is very vague and this has already been done in the paper's abstract.

Re: Thanks for your valuable comments. We carefully describe the all methods in detail in the Methods and abstract sections.

Co15: Make, model, city and country of manufacture must be entered for all equipment used

Re: Thank you for your valuable comment. We add the Make, model, city and country of manufacture for all equipment used.

Co16: Data processing should be detailed in more detail, how were the data processed? Were they normalized? How were repeated measurements analyzed over time? In relation to regression analysis? How was significance chosen? Based on the correlation coefficient? .... The statistical model must be entered

Re: We appreciate your insight and have revised the section of the Data processing and analysis. The data were first processed by Microsoft Excel 2016. Each individual bottle was an experimental unit for all collected data. All data are measured values, not standardized and were presented as averages. We first processed one-way ANOVA on our date, and then by Duncan's multiple range test evaluate the differences among treatments. Meanwhile, regression (REG) analysis was conducted to evaluate the linear and quadratic effects of the increasing levels of RDS on the fermentation parameters. significance chosen based on Duncan's multiple range test (Line 176-182).

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have made a profound improvement to the manuscript. I recommend accepting the paper.