The Effect of Lignin Composition on Ruminal Fiber Fractions Degradation from Different Roughage Sources in Water Buffalo (Bubalus bubalis)

Round 1

Reviewer 1 Report

Dear authors,

this is to my opinion an overall well-written paper and it contains highly important and topical information. I have a few major and minor suggestions that you should consider while preparing the revision of your paper. Details are listed in the following:

In general, please thoroughly revise English style and syntax; I recommend you to use a professional English editing service.

Abstract: L23, investigating the classes of lignin monomers or their composition? Please revise. L26, . . . cellulose (CEL), and . . . L30, . . . is negatively correlated . . . There are a lot of such things in the manuscript, so, please revise English! L32, if you give R², please also give the corresponding p-value.

Introduction: L68-69, this is contradictory as it is written here; if maturity - lignification - S-lignin are increasing, this should decrease digestibility. L71-72, again, seems contradictory to your own findings . . . you explained it in the discussion section, but here the reader could become confused. L73-80, rewrite this section, provide clear hypotheses or objectives that derive from literature discussion above. You introduced lignin composition effects on fiber digestibility, here, you suddenly speak about feed selection by buffalos.

Materials and methods: L85, better Total Mixed Ration (TMR). L87-89, . . . were used. Delete the rest of this sentence, it doesn't matter here. L99, please clearly indicate whether NDF and ADF are pre-treated with amylase (aNDF) and expressed exclusive of residual ash (aNDFom, ADFom). L103-115, give references for the equations. L110, Dr is a starnge abbreviation for digestibility. DNA sequencing is not mentioned in the abstract! L130-131, . . . were bred in . . . doesn't matter, please delete. L131, what is jejunitas? Please use correct English wording! L158-167, should appear at the end of section 2.4. Why didn't you use qiime2 and ASVs instead of qiime and OTUs? Please be much more specific on statistical analysis! Sound statistical analysis of your data is very important and you must describe how you did it as precise as possible! Add, e.g., descriptions of the comparisons of degradation parameters and ED, and descriptions of the correlation analysis. For ED calculation, which passage rate did you assume? Revise the tables! Tab. 7, better write p < 0.001, not p = 0.000.

Supplemental material: I miss information on sequencing quality. Please provide rarefaction curves and OTU tables as supplements. You should provide the rare data files in a public repository and give the accession number in the paper.

Author Response

Reviewer 1:

This is to my opinion an overall well-written paper and it contains highly important and topical information. I have a few major and minor suggestions that you should consider while preparing the revision of your paper. Details are listed in the following:

Response: Dear reviewer, thanks for your professional advices. We have fully considered your comments and responded these comments point by point. We have tried our best to revise our manuscript according to the comments.

1.In general, please thoroughly revise English style and syntax; I recommend you to use a professional English editing service.

Response: Dear reviewer, thanks for your suggestions. We have revised English style and syntax. Some improper English expressions have been corrected.

2. L23, investigating the classes of lignin monomers or their composition? Please revise.

Response: According to your advice, we have rewritten the abstract. This sentence has been revised to “investigate the relation between the lignin classes’ coniferyl alcohol (G), ρ-coumaryl alcohol (H), and sinapyl alcohol (S) and ruminal fiber degradability in water buffalo.”

3. L26, . . . cellulose (CEL), and . . . L30, . . . is negatively correlated . . . There are a lot of such things in the manuscript, so, please revise English!

Response: Thanks for your suggestion, we have revised.

4. L32, if you give R², please also give the corresponding p-value.

Response: Thanks for your suggestion, we have added the p-value in the abstract following R².

5. L68-69, this is contradictory as it is written here; if maturity - lignification - S-lignin are increasing, this should decrease digestibility. L71-72, again, seems contradictory to your own findings . . . you explained it in the discussion section, but here the reader could become confused.

Response: Thanks for this comment, we revised the section. The revised section is as follow “Although lignin is considered a critical limiting factor for cell-wall digestibility, the variations in cell-wall digestibility cannot be attributed solely to lignin content. Besides the lignin concentration, monomer composition was a critical factor affecting fiber degradation in animal digestibility studies. However, few trials have been evaluated the contribution of lignin types in altering forage utilization. During forage maturation, lignin composition in the cell-wall shifts from G-type to S-type was identified as a typical marker of cell-wall maturation. A previous study had reported that a lower S/G ratio in a Populus family was linked to a higher extent of xylose released by diluted acid hydrolysis. Jung tested various forage maize and also proved that lower S monomer components were mainly related to more milk and meat production. Through previous studies using Arabidopsis mutant, transgenic alfalfa, and biomimetic cell wall model, it was reported that G-type and S-type lignin could alter cell-wall digestibility by affecting cellulolytic enzymes activity and rumen microbes action. However, Fontaine put forward a completely different opinion that the S-type content is positive correlation with maize digestibility. In Arabidopsis plant tissues, the abundance of S-type lignin plays a more important role in glucose production.”

6. L73-80, rewrite this section, provide clear hypotheses or objectives that derive from literature discussion above. You introduced lignin composition effects on fiber digestibility, here, you suddenly speak about feed selection by buffalos.

Response: Dear reviewer, buffaloes live in the tropical area zone which is characterized by elevated temperatures and high-fiber and low-protein roughage supply, such as rice straw and field crop production. Hence, research the effective of lignin composition to fiber digestibility can establish a new strategy for buffalo roughage selection. Meanwhile, we have rewritten the section, add hypotheses derive from literature discussion. The revised section is as follow “Worldwide, there are abundant crop by-products, including different kinds of roughages that vary in lignin monomer structures. However, few reports discuss the composition of the lignin monomer and its linkage to roughage utilization. The present study examines the role of lignin monomer classes on ruminal fiber fractions degradability in water buffalo, using different roughage sources, which aimed to establish a novel approach to evaluate roughage's nutritional value. Thus we hypothesized that the lignin monomer composition is a leading marker affecting fiber fractions degradability of roughage.”

7. L85, better Total Mixed Ration (TMR).

Response: In accordance with your suggestion, we have corrected those mistakes in the revised manuscript.

8. L87-89, . . . were used. Delete the rest of this sentence, it doesn't matter here.

Response: Dear reviewer, according to your suggestion, we have deleted the rest of the sentence, and exchange the sequences in the material and methods.

9. L99, please clearly indicate whether NDF and ADF are pre-treated with amylase (aNDF) and expressed exclusive of residual ash (aNDFom, ADFom).

Response: Thanks for your advice, we ensure we used amylase before determine NDF. We have expressed exclusive of residual ash (aNDFom, ADFom) and used the method by AOAC.[1]

1. Science, A.F. Use of detergent system terminology and criteria for submission of manuscripts on new , or revised , analytical methods as well as descriptive information on feed analysis and / or variability. 2005, 118, 181–186, doi:10.1016/j.anifeedsci.2004.11.011.
2. L103-115, give references for the equations.

Response: Dear reviewer, in the new version, we have given references for the equations.[2][3]

2. P.H. ROBINSON, J.G.F. and S.T. EVALUATION OF MATHEMATICAL MODELS TO DESCRIBE NEUTRAL DETERGENT RESIDUE IN TERMS OF ITS SUSCEPTIBILITY TO DEGRADATION IN THE RUMEN. Anim. Feed Sci. Technol. 1986, 15, 249–271.
3. By, E.R.; Mcdonald, I. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 1979, 499–503.
4. L110, Dr is a strange abbreviation for digestibility. DNA sequencing is not mentioned in the abstract!

Response: According to your suggestion, Dr means degradation rate, this abbreviation refer to [4]. What’s more, we have rewritten the abstract, DNA sequencing is mentioned in the revised publication.

4. Xu, Q.; Zhong, H.; Zhou, J.; Wu, Y.; Ma, Z.; Yang, L.; Wang, Z.; Ling, C.; Li, X. Lignin degradation by water buffalo. Trop. Anim. Health Prod. 2021, 53, 1–8, doi:10.1007/s11250-021-02787-z.
5. L130-131, . . . were bred in . . . doesn't matter, please delete.

Response: According to your suggestion, we have deleted parts of the sentence in the revised manuscript.

13. L131, what is jejunitas? Please use correct English wording!

Response: Thanks for your suggestion, and we use “fasting” instead of “jejunitis” in the revised manuscript.

14. L158-167, should appear at the end of section 2.4.

Response: Thanks for your suggestion, we have moved the section at the end of section 2.4.

15. Why didn't you use qiime2 and ASVs instead of qiime and OTUs?

Response: Dear reviewer, we admit that qiime2 and ASVs is advanced. But when we did this experiment, qiime2 and ASVs hadn’t been invented. Meanwhile, I think using qiime and OTUs to analysis our datas is reliable enough and convincing, refer to [5–7].

5. Ma, X.; Li, X.; Liu, J.; Cheng, Y.; Zou, J.; Zhai, F.; Sun, Z.; Han, L. Soil microbial community succession and interactions during combined plant/white-rot fungus remediation of polycyclic aromatic hydrocarbons. Sci. Total Environ. 2021, 752, 142224, doi:10.1016/j.scitotenv.2020.142224.
6. Shi, X.; Zhao, X.; Ren, J.; Dong, J.; Zhang, H.; Dong, Q.; Jiang, C.; Zhong, C.; Zhou, Y.; Yu, H. Influence of Peanut, Sorghum, and Soil Salinity on Microbial Community Composition in Interspecific Interaction Zone. Front. Microbiol. 2021, 12, 1–13, doi:10.3389/fmicb.2021.678250.
7. Luan, L.; Jiang, Y.; Cheng, M.; Dini-Andreote, F.; Sui, Y.; Xu, Q.; Geisen, S.; Sun, B. Organism body size structures the soil microbial and nematode community assembly at a continental and global scale. Nat. Commun. 2020, 11, 1–11, doi:10.1038/s41467-020-20271-4.
8. Please be much more specific on statistical analysis! Sound statistical analysis of your data is very important and you must describe how you did it as precise as possible! Add, e.g., descriptions of the comparisons of degradation parameters and ED, and descriptions of the correlation analysis. For ED calculation, which passage rate did you assume?

Response: Dear reviewer, thanks for your advice. According to your suggestion, we have revised the statistical analysis. The revised section is as follow “We analyzed the chemical composition and degradation characteristics of samples using a t-test with SPSS 19.0. Multiple regression prediction model y = a + b₁x₁ + b₂x₂ + b₃x₃ + ··· + b_mx_m, where y is NDFD, ADFD, CELD, HCD and ADLD, a is the random error constant, b is the independent variable coefficient, x is ADL content, S / G ratio, H / G ratio, H / S ratio, H content, s content, G content, (G + S + H) content, G / (G + S + H) ratio, S / (G + S + H) ratio, H / (G + S + H) ratio, (G + S) / (G + S + H) ratio, (G + H) / (G + S + H) ratio and (H + S) / (G + S + H) ratio, the regression equation was obtained by stepwise regression of the above model variables with SPSS 19.0. Statistical significance was set at p < 0.05 (significant difference) and p < 0.01 (extremely significant difference). ”

And for ED, we have mentioned in 2.2. Sample Analyses. ED = a + b × c / (c + k). where a is the fast degradation part of the tested feed (%), b is the slow degradation part of the tested feed (%), c is the degradation rate of the slow degradation part of tested feed (h⁻¹), and k is the rumen flow rate of feed to be tested (h⁻¹) (0.025/h⁻¹ was adopted in this test).

17. Revise the tables! Tab. 7, better write p < 0.001, not p = 0.000.

Response: Dear reviewer, the p=0.000 has been revised to p < 0.01.

18. Supplemental material: I miss information on sequencing quality. Please provide rarefaction curves and OTU tables as supplements. You should provide the rare data files in a public repository and give the accession number in the paper.

Response: Dear reviewer, we have provided the rare data files in NCBI, and the accession numbers are: SRR15970504; SRR15970503; SRR15970502; SRR15970501; SRR15970500; SRR15970499. And we have given them in the paper. OUT tables and shannon curves are as follow. For the shannon curves, there are 24 samples curves in a year (6 samples per season) in this figure. However, we did the experiment in spring, so we used 6 samples of spring in this paper.

Author Response File: Author Response.docx

Reviewer 2 Report

To Authors

The research undertaken at work is interesting and innovative, but I believe that the manuscript still needs some fine-tuning.

Title

I suggest changing the title to one that is more adequate to the research performed and the authors' expectations.

Line 43: Lignin is not a phenolic compound, but a polymer whose monomers are organic compounds derived from phenolic alcohols. Please do not generalize the concepts.

Line 44: ... It increases with plant maturity ... - please construct a sentence correctly and relate your thoughts to the level of lignin. This is too much of a generalization in the current release.

Introduction

Line 48-49: Please briefly elaborate on the interesting topic of lignin digestibility in buffalo stomachs and the uniqueness of the research conducted. This is valuable information.

Line 73-80: There is no research hypothesis in publication. What is the research hypothesis of this manuscript? Please create a hypothesis to find out what the Authors wanted to check? The formulation of a research hypothesis is an essential part of a scientific study. In order for it to be possible to try or precisely formulate a research hypothesis, it is necessary to define a research problem, i.e. a question that defines the dimension and quality of the scope of knowledge that we want to acquire and the purpose of creating a scientific work.

Material and methods

Line 82: We do not put a period in the titles of chapters and subsections. Applies to the entire publication.

Line 83-85: Despite a very interesting research idea, I believe that 3 animals is not enough to draw any meaningful conclusions. Moreover, isn't the age of 5 too much for experimental animals?

Line 87-97: Please keep a logical chronology in this part of the methodology. First describe the animals, then the feed, and then inform what laboratory analyzes were made. It does not find out when the animals received a particular type of feed. Was animal feed separated and digestibility analyzed, or was the feed administered ad libitum?

Line 99: Which attempts were you analyzing? Please specify.

Line 117: Please specify in the methodology about which the authors write. The reader must guess this.

Line 133: Please specify the exact manufacturer of the test for DNA extraction from microorganisms.

Line 172: Which statistical program was used?

Tables

There is no description of the statistical calculations under the tables. I did not find information what the letters from a-f mean, and what is ED?

Conclusions

Line 357-364: Please precisely specify the conclusions of the work. And most importantly, what is the application nature of the research? Can the Authors define it?

Author Response

Reviewer 2:

The research undertaken at work is interesting and innovative, but I believe that the manuscript still needs some fine-tuning.

Response: Dear reviewer, thanks for your professional advices. We have fully considered your comments and responded these comments point by point. We have tried our best to revise our manuscript according to the comments.

1. I suggest changing the title to one that is more adequate to the research performed and the authors' expectations.

Response: Dear reviewer, according to your suggestion, the title “Fiber Fractions Degradation in Buffalo Rumen in Response to Lignin Composition from Different Roughages” have been changed to “The Effect of Lignin Fractions and Composition on Ruminal Fiber Degradability from Different Roughage Sources in Water Buffalo (Bubalus bubalis).”

2. Line 43: Lignin is not a phenolic compound, but a polymer whose monomers are organic compounds derived from phenolic alcohols. Please do not generalize the concepts. Line 44: ... It increases with plant maturity ... - please construct a sentence correctly and relate your thoughts to the level of lignin. This is too much of a generalization in the current release.

Response: Dear reviewer, thanks for your suggestion. This section has been revised to “Lignin is a complex aromatic polymer in the cell wall, which increasing with the plant maturity development. It consists of phenylpropane units, filling the gaps between cel-lulose and hemicellulose likes a resin. In addition, it is known that lignin content pro-vides a structural strength and rigidity for plant cell walls.”

3. Line 48-49: Please briefly elaborate on the interesting topic of lignin digestibility in buffalo stomachs and the uniqueness of the research conducted. This is valuable information.

Response: Dear reviewer, we rich the section of lignin digestibility in buffalo stomachs, the revised section is as follow “Few animal species have been reported with the ability of lignin degradation except termites and longicorn, interestingly, recently buffalo have been found to have the ability to degrade lignin. Xu and Zhong found that when ingesting the same forage with high fiber and low protein, buffalo showed higher dry mater intake and stronger growth performance than cattle. through comparative experiments between buffalo and cattle, they reported that buffalo could degrade lignin efficiently but cattle not.”

4. Line 73-80: There is no research hypothesis in publication. What is the research hypothesis of this manuscript? Please create a hypothesis to find out what the Authors wanted to check? The formulation of a research hypothesis is an essential part of a scientific study. In order for it to be possible to try or precisely formulate a research hypothesis, it is necessary to define a research problem, i.e. a question that defines the dimension and quality of the scope of knowledge that we want to acquire and the purpose of creating a scientific work.

Response: Dear reviewer, we have rewritten the section, add research hypotheses of this manuscript. The revised section is as follow “Worldwide, there are abundant crop by-products, including different kinds of roughages that vary in lignin monomer structures. However, few reports discuss the composition of the lignin monomer and its linkage to roughage utilization. The present study examines the role of lignin monomer classes on ruminal fiber fractions degradability in water buffalo, using different roughage sources, which aimed to establish a novel approach to evaluate roughage's nutritional value. Thus we hypothesized that the lignin monomer composition is a leading marker affecting fiber fractions degradability of roughage.”

5. Line 82: We do not put a period in the titles of chapters and subsections. Applies to the entire publication.

Response: According to your suggestion, we have revised in the manuscript.

6. Line 83-85: Despite a very interesting research idea, I believe that 3 animals is not enough to draw any meaningful conclusions. Moreover, isn't the age of 5 too much for experimental animals?

Response: Dear reviewer, thanks for your suggestions. In our experiment, all the experimental animals were in the same condition (health, comfortable and free from external interference). Meanwhile, for nylon bag technique, 3 Bovine animals are enough, refers to [8,9]. In addition, water buffaloes have a remarkably long productive lifespan : 6 - 8 calving (9 - 11 years of life) were reported for Italian buffaloes, and 12 - 14 calving (15 - 17 years) for water buffaloes in Brazil, refers to [10,11]. Hence the age of 5 is suitable for buffaloes.

8. Liu, Y.; Li, R.; Wu, H.; Meng, Q.; Khan, M.Z.; Zhou, Z. In vitro neutral detergent cellulase method and chemical composition to predict in vivo fermentable organic matter of roughages. Animals 2021, 11, doi:10.3390/ani11061594.
9. Ali, M.; Cone, J.W.; van Duinkerken, G.; Klop, A.; Blok, M.C.; Bruinenberg, M.; Khan, N.A.; Hendriks, W.H. Variation between individual cows in in situ rumen degradation characteristics of maize and grass silages. NJAS - Wageningen J. Life Sci. 2016, 78, 167–173, doi:10.1016/j.njas.2016.05.009.
10. Seibt, K.D.; Häussler, S.; Vecchio, D.; Decarlo, E.; Ceciliani, F.; Sauerwein, H. Comparison of telomere lengths in leukocytes and in nasal and vaginal epithelial cells from Water Buffaloes (Bubalus bubalis) of different ages. Res. Vet. Sci. 2019, doi:10.1016/j.rvsc.2019.04.013.
11. Galeazzi, P.M.; Mercadante, M.E.Z.; Silva, J.A. Genetic parameters for stayability in Murrah buffaloes. 2010, 252–256, doi:10.1017/S0022029910000075.

7. Line 87-97: Please keep a logical chronology in this part of the methodology. First describe the animals, then the feed, and then inform what laboratory analyzes were made.

Response: Thanks for your professional suggestions, we revise the logical mistake. In the revised manuscript, we have changed the description sequence of material and method section.

8. It does not find out when the animals received a particular type of feed. Was animal feed separated and digestibility analyzed, or was the feed administered adlibitum?

Response: The animals received a Total Mixed Ration (TMR), it’s mentioned in 2.1.

The contents and nutrient contents of TMR as follow:

9. Line 99: Which attempts were you analyzing? Please specify.

Response: Thanks for your advice, we have revised it into “In order to analyze the fiber content in roughages. Eight types of roughage (rice straw, bracts, maize stalks, peanut vine, sweet potato vine, rice husk, maize silage, vinasse) were analyzed for the neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose (CEL), and hemicellulose (HC) based on the method revised by AOAC, HC and CEL were estimated respectively as NDF - ADF, and ADF - ADL. Samples were pretreated with α-amylase before NDF determination.” Refer to [1]

1. Science, A.F. Use of detergent system terminology and criteria for submission of manuscripts on new , or revised , analytical methods as well as descriptive information on feed analysis and / or variability. 2005, 118, 181–186, doi:10.1016/j.anifeedsci.2004.11.011.
2. Line 117: Please specify in the methodology about which the authors write. The reader must guess this.

Response: Dear reviewer, the method of Py-GC-MS means Pyrolysis-gas chromatography/mass spectrometry. And we add eight roughages’ name. The revised sentence is as follow “Eight types of roughage (rice straw, bracts, maize stalks, peanut vine, sweet potato vine, rice husk, maize silage, vinasse) were analyzed for lignin composition by Py-GC-MS. 4.25 mg sample of each roughage was placed in a platinum cup on a FRONTIER 3030D pyrolyzer, and pyrolysis temperature was adjusted to 500 ℃.”

11. Line 133: Please specify the exact manufacturer of the test for DNA extraction from microorganisms.

Response: Dear reviewer, the experiment of DNA extraction from microorganisms was done by ourselves.

12. Line 172: Which statistical program was used?

Response: Dear reviewer, the statistical program we used was SPSS 19.0. And we have revised in the paper. The revised section is as follow “We analyzed the chemical composition and degradation characteristics of samples using a t-test with SPSS 19.0. Multiple regression prediction model y = a + b₁x₁ + b₂x₂ + b₃x₃ + ··· + b_mx_m, where y is NDFD, ADFD, CELD, HCD and ADLD, a is the random error constant, b is the independent variable coefficient, x is ADL content, S / G ratio, H / G ratio, H / S ratio, H content, s content, G content, (G + S + H) content, G / (G + S + H) ratio, S / (G + S + H) ratio, H / (G + S + H) ratio, (G + S) / (G + S + H) ratio, (G + H) / (G + S + H) ratio and (H + S) / (G + S + H) ratio, the regression equation was obtained by stepwise regression of the above model variables with SPSS 19.0. Statistical significance was set at p < 0.05 (significant difference) and p < 0.01 (extremely significant difference).”

13. There is no description of the statistical calculations under the tables. I did not find information what the letters from a-f mean, and what is ED?

Response: Dear reviewer, we are sorry that we just added the notes about letters from a-f mean in Table 5. And now we have added the notes in Table 1 to 5. And letters from a-f mean “The different letters on the shoulder means significant difference (p<0.05), the same letters mean no significant difference (p>0.05).” What’s more, the ED we have explained in 2.2. Sample Analyses. I guess you missed it by accident.

14. Line 357-364: Please precisely specify the conclusions of the work. And most importantly, what is the application nature of the research? Can the Authors define it?

Response: Dear reviewer, thanks for your professional suggestions, we have revised the conclusion in the paper. The details are as follows:

“To summaries, it was concluded that there is a strong correlation between lignin monomer composition and fiber fractions degradation in roughage, which NDFD=0.647-0.071 * H / S (R²=0.628, p <0.01), ADFD=0.427-0.045*H/S+0.826*S/G (R²=0.678, p<0.01), ADLD=0.161-1.918*H+3.152*S (R²=0.758, p <0.01), CELD=0.724-0.081*H/S (R²=0.620, p<0.01), HCD=0.522-0.038*H/S+0.649*S/G (R²=0.709, p<0.01). The relationship between monomer composition change based on the S-type lignin monomer and the determination of biodegradable fiber fractions degradation was much closer. Moreover, we found that the capacity to degrade lignin of buffalo which based on its unique rumen microorganisms, the degradation extent related to the H-type monomer. The study provides a new method for evaluating the value of roughage, reducing fiber fractions loss and improving ruminants' production performance of ruminants.”

Author Response File: Author Response.docx

Reviewer 3 Report

General comments:

The manuscript needs some corrections.

1.    I suggest that the authors determine an objective that involves the general idea of the manuscript and all the analyzes performed.

2.    The abstract needs to be rewrite.

3.    The introduction needs to add information that involves the analyzes carried out.

4.    The material and methods and results topics need to follow a chronological or methodological order. Try to make the manuscript easy to read by connecting ideas and topics.

5.    The discussion topic has a revision style. The topic of discussion is the area where the authors biologically explain their results. You presented many results and all of them important. However, a discussion of most of the results was not observed; only a review or comparison of the data was observed.

Abstract:

At this point, the experiment is unclear about the objective of the experiment. There is a lot of information but there is no connection between the ideas. So it is difficult to understand if the objective is to evaluate the components of the lignin, or feeds? Models Fiber fractions? Or did you make correlations? Rewrite to make it clear.

You should be add relevant information and avoid lines that no increase the relevance of the abstract (E.g. lines 38-39).

Line (23): Add average age, body weight, sex, and breed of the animals. 

Line (23): Add the experimental design.

Line (23): clarify the treatments of the study.

Line (31): fitting degree? According to? Akaikes index? Correlation coefficients of concordance? etc. Detail.

Line (32-39): What is the conclusion? If the authors do not determine the real objective, they will not know how to describe the correct results or the correct conclusion. At this point, the objective is different from the results and the conclusion.

Keywords: delete: ADL and rumen

Introduction:

Line (43): Delete “main”.

Line (77): Add the experimental hypothesis. 

Line (78-79): Delete the second “on fiber fractions degradation”.

Material and methods:

Line (83-84): Add average body weight and breed of the animals.

Line (116): Describe a full name

Line (168): What is KECG?

Line (172-173): Were all parameters evaluated with regression analysis? In the abstract you report models, what models? How did you carry out the analysis of the effectiveness of the models? Correlations? What is the statistical regression model used? What is experimental design? Add all statistical information.

Results and discussion

Improve the resolution of all figures.

Line (221): Delete “The composition … … Figure 2.”

Line (232-234): Delete this lines

Line (234-236): These lines belong to the topic of discussion. Write here the specific results of the figure 3.

Line (266-290): I don’t understand the idea of these paragraphs. Only lines 279-286 are relevant to me. Besides, these lines could be placed into the results topic.

Line (275-277): What is the purpose of this idea? I consider it irrelevant

Line (295): What is meaningful "linear structure"? Describe

Line (325-326): This idea is repetitive. You wrote these lines in other words many times throughout the text.

Tables: Add footnotes to describe short names or initials like NDFD. Tables should be self-explanatory.

Table 8: What is rumen1, rumen2, rumen3 …?.

Figure 1: Add signs where there are significant differences and improve the figure.

Conclusion:

Lines (357-364): Rewrite so this text is better explained, less confusing and according to the manuscript objective.

Author Response

Reviewer3

General comments:

The manuscript needs some corrections.

1. I suggest that the authors determine an objective that involves the general idea of the manuscript and all the analyzes performed.
2. The abstract needs to be rewrite.
3. The introduction needs to add information that involves the analyzes carried out.
4. The material and methods and results topics need to follow a chronological or methodological order. Try to make the manuscript easy to read by connecting ideas and topics.
5. The discussion topic has a revision style. The topic of discussion is the area where the authors biologically explain their results. You presented many results and all of them important. However, a discussion of most of the results was not observed; only a review or comparison of the data was observed.

Response: Dear reviewer, thanks for your professional advices. We have revised the paper according to your suggestion.

Abstract:

1. At this point, the experiment is unclear about the objective of the experiment. There is a lot of information but there is no connection between the ideas. So it is difficult to understand if the objective is to evaluate the components of the lignin, or feeds? Models Fiber fractions? Or did you make correlations? Rewrite to make it clear.

You should be add relevant information and avoid lines that no increase the relevance of the abstract (E.g. lines 38-39).

Response: Dear reviewer, thanks for your suggestions, we have rewritten the abstract as follow:

Water buffalo (Bubalus bubalis), known for its unique utilization of low-quality fibrous feeds and out-standing digestion performance, highlighting its role as an animal model in studying the relation between fiber fractions and digestibility efficiency. Among fiber fractions, the lignin classes attracted wide atten-tion through ruminant nutrition studies, especially that it varies greatly among different feedstuffs af-fecting feed intake and animal digestibility. Therefore, the present study aims to investigate the relation between the lignin classes’ coniferyl alcohol (G), ρ-coumaryl alcohol (H), and sinapyl alcohol (S) and ruminal fiber degradability in water buffalo. Hence, three female water buffaloes (Neri Raffi × Mediterranean, five years old , 480 ± 20 kg) were assigned for an in-vivo study by the nylon-bag method, ex-amining eight kinds of roughage (rice straw, bracts, corn stalks, peanut vine, sweet potato vine, rice husk, maize silage, vinasse). All the experimental roughage types were analyzed for the effective degradability (ED) of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), cellulose (CEL), and hemicellulose (HC) fractions. Also, lignin degradation bacteria were isolated, and DNA was extracted for further classification. Then, a prediction model for the roughage fiber degradation was established; based on the characteristics of lignin monomer content. The results showed that S, S / G, S / (G + S + H) correlated positively with the determined parameters, however ; H / S, (G + H) / (G + S + H) was negatively correlated, except for ADLD (effective degradability of ADL). The model with the highest fitting degree was HCD (effective degradability of HC) = 0.522 - 0.038 × H / S + 0.649 × S / G (R²=0.709, p<0.01), except ADLD. These results indicated that lignin monomer composition is closely related to the utilization rate of roughage fiber. S-type lignin monomer plays a vital role in the fiber degradation of roughage. The more S-type lignin monomer content, the higher rumen degradation of biodegradable fiber composition, therefore the establishment of a prediction model also was based on S-type lignin monomer change. Also, the microbial ruminal community was dominated by Prevotella_1, followed by 226, Rikenellaceae_RC9_gut_group, Ruminococcaceae_UCG-011 Fibrobacter, Ruminococcus_1, 227 and Saccharofermentans. The experiment provides a scientific basis for building a new technological method using lignin composition to evaluate buffalo roughage. In addition, this experiment also revealed that buffalo owns rumen microorganisms with lignin degradation potential.

2. Line (23): Add average age, body weight, sex, and breed of the animals.

Line (23): Add the experimental design.

Line (23): clarify the treatments of the study.

Response: According to your suggestion, we have added average age, body weight, sex, and breed of the animals and the experimental design. And we clarify the treatments of the study. The revised sentences are as follow “three female water buffaloes (Neri Raffi × Mediterranean, five years old , 480 ± 20 kg) were assigned for an in-vivo study by the nylon-bag method, examining eight kinds of roughage (rice straw, bracts, corn stalks, peanut vine, sweet potato vine, rice husk, maize silage, vinasse). All the experimental roughage types were analyzed for the effective degradability (ED) of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), cellulose (CEL), and hemicellulose (HC) fractions. Also, lignin degradation bacteria were isolated, and DNA was extracted for further classification. Then, a prediction model for the roughage fiber degradation was established; based on the characteristics of lignin monomer content.”

3. Line (31): fitting degree? According to? Akaikes index? Correlation coefficients of concordance? etc. Detail.

Response: Dear reviewer, we have revised it and added the p-value (R²=0.709, p<0.01)

4. Line (32-39): What is the conclusion? If the authors do not determine the real objective, they will not know how to describe the correct results or the correct conclusion. At this point, the objective is different from the results and the conclusion.

Response: Dear reviewer, we have revised the conclusion in abstract, the revised conclusion is as follow “The experiment provides a scientific basis for building a new technological method using lignin composition to evaluate buffalo roughage. In addition, this experiment also revealed that buffalo owns rumen microorganisms with lignin degradation potential.”

5. Keywords: delete: ADL and rumen

Response: Dear reviewer, we have deleted ADL and rumen in the keywords.

Introduction:

6. Line (43): Delete “main”.

Response: According to your suggestion, we have corrected those mistakes in the revised manuscript.

7. Line (77): Add the experimental hypothesis. Line (78-79): Delete the second “on fiber fractions degradation”.

Response: Dear reviewer, we have added the experimental hypothesis, and we rewrite the section. The section is as follow “Worldwide, there are abundant crop by-products, including different kinds of roughages that vary in lignin monomer structures. However, few reports disscuss the composition of the lignin monomer and its linkage to roughage utilization. The pre-sent study aimed to examine the role of lignin monomer composition on ruminal fiber fractions degradability in water buffalo, using different roughage sources, which may establish a novel approach to evaluate roughage's nutritional value. Thus we hy-pothesized that the lignin monomer composition is a leading marker affecting fiber fractions degradability of roughage.”

Material and methods:

8. Line (83-84): Add average body weight and breed of the animals.

Response: According to your suggestion, we have added average body weight and breed of the animals in the revised manuscript.

9. Line (116): Describe a full name

Response: According to your suggestion, we have described a full name Pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS)

10. Line (168): What is KECG?

Response: Dear reviewer, we guess you refer the KEGG, which full name is Kyoto Encyclopedia of Genes and Genomes, KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies.

11. Line (172-173): Were all parameters evaluated with regression analysis? In the abstract you report models, what models? How did you carry out the analysis of the effectiveness of the models? Correlations? What is the statistical regression model used? What is experimental design? Add all statistical information.

Response: Dear reviewer, thanks for your advice. According to your suggestion, we have revised the statistical analysis. The revised section is as follow “We analyzed the chemical composition and degradation characteristics of samples using a t-test with SPSS 19.0. Multiple regression prediction model y = a + b₁x₁ + b₂x₂ + b₃x₃ + ··· + b_mx_m, where y is NDFD, ADFD, CELD, HCD and ADLD, a is the random error constant, b is the independent variable coefficient, x is ADL content, S / G ratio, H / G ratio, H / S ratio, H content, s content, G content, (G + S + H) content, G / (G + S + H) ratio, S / (G + S + H) ratio, H / (G + S + H) ratio, (G + s) / (G + S + H) ratio, (G + H) / (G + S + H) ratio and (H + s) / (G + S + H) ratio, the regression equation was obtained by stepwise regression of the above model variables with SPSS 19.0. Statistical significance was set at p < 0.05 (significant difference) and p < 0.01 (extremely significant difference). ”

Results and discussion

12. Improve the resolution of all figures.

Response: Dear reviewer, thanks for your advice, we have replaced the original figures with high-resolution figures.

13. Line (221): Delete “The composition … … Figure 2.”

Response: Dear reviewer, we have deleted it according to your suggestion.

14. Line (232-234): Delete this lines

Response: Dear reviewer, we have deleted it according to your suggestion.

15. Line (234-236): These lines belong to the topic of discussion. Write here the specific results of the figure 3.

Response: Dear reviewer, according to your advice, this section is revised as follow “As shown in Figure 3. The rumen flora is enriched in 24 sorts of COG functions. The top three are general function prediction only (R), transcription (K) and carbohydrate transport and metabolism (G). The last four with significantly less are cytoskeleton (Z), chromatin structure and dynamics (B), RNA processing and modification (A) and extracellular structures (W). ”

16. Line (266-290): I don’t understand the idea of these paragraphs. Only lines 279-286 are relevant to me. Besides, these lines could be placed into the results topic.

Response: Dear reviewer, thanks for your advice, this paragraph has been written in the corresponding result.

17. Line (275-277): What is the purpose of this idea? I consider it irrelevant

Response: Dear reviewer, thanks for your advice, after our consideration, we thought the description here was really inappropriate, so we revised it into “In the present study, the selected eight kinds of roughage were by-products and silage either for monocotyledonous crops (rice straw, bracts, and corn stalks) or dicotyledonous crops (peanut vine and sweet potato vine, the rest of rice husk, maize silage, and vinasse). Although the monocotyledons and dicotyledons include the content from S-type, G-type, and H-type lignin, the monocotyledon plants tend to be more abundant in H-type lignin monomers”

18. Line (295): What is meaningful "linear structure"? Describe

Response: Dear reviewer, linear structure is one of the geometric forms of macromolecular chains. The whole molecule is like a long chain. Polymer with linear structure has good elasticity and plasticity. So, S-type lignin can extend to the secondary wall of the plant cell wall.

19. Line (325-326): This idea is repetitive. You wrote these lines in other words many times throughout the text.

Response: Thanks for your professional advice. This sentence has been changed to that “Lignin is a structure stability polymer, that has not been reported that mammals can degrade and utilize lignin.”

20. Tables: Add footnotes to describe short names or initials like NDFD. Tables should be self-explanatory.

Response: Dear reviewer, thanks for your suggestion, we have added footnotes to describe short names in the paper.

21. Table 8: What is rumen1, rumen2, rumen3 …?.

Response: Dear reviewer, it is our mistake. The correct wordings are Rumen0.1, Rumen0.2, Rumen0.3…which means different rumen liquid samples from buffalo rumen. A total of six samples were from three buffalo.

22. Figure 1: Add signs where there are significant differences and improve the figure.

Response: Thanks for your suggestion, according to your advice, we have add significant differences in the figure 1. Meanwhile, we improve the quality of figure 1.

Conclusion:

23. Lines (357-364): Rewrite so this text is better explained, less confusing and according to the manuscript objective.

Response: Dear reviewer, thanks for your professional suggestions, we have revised the conclusion in the paper. The details are as follows:

“To summaries, it was concluded that there is a strong correlation between lignin monomer composition and fiber fractions degradation in roughage, which NDFD=0.647-0.071 * H / S (R²=0.628, p <0.01), ADFD=0.427-0.045*H/S+0.826*S/G (R²=0.678, p<0.01), ADLD=0.161-1.918*H+3.152*S (R²=0.758, p <0.01), CELD=0.724-0.081*H/S (R²=0.620, p<0.01), HCD=0.522-0.038*H/S+0.649*S/G (R²=0.709, p<0.01). The relationship between monomer composition change based on the S-type lignin monomer and the determination of biodegradable fiber fractions degradation was much closer. Moreover, we found that the capacity to degrade lignin of buffalo which based on its unique rumen microorganisms, the degradation extent related to the H-type monomer. The study provides a new method for evaluating the value of roughage, reducing fiber fractions loss and improving ruminants' production performance of ruminants.”

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear authors. Thanks for the revision. I think the manuscript has become much better. Please revise the references 19-21! For example, ref. 19 was written by Peter Uden et al., not by A.F. Science . . . this is a funny mistake, it should, however, corrected before publication. Please carefully check the rest of the manuscript for such "minor" errors. Good luck with the manuscript!

Author Response

Reviewer 1

Dear authors. Thanks for the revision. I think the manuscript has become much better. Please revise the references 19-21! For example, ref. 19 was written by Peter Uden et al., not by A.F. Science . . . this is a funny mistake, it should, however, corrected before publication. Please carefully check the rest of the manuscript for such "minor" errors. Good luck with the manuscript!

Response: Dear reviewer, thanks for your advice. The reference 19 has been revised. And we have carefully checked the rest of the paper.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear Authors, thank you for your answers. Unfortunately, the publication still needs to be corrected. First, I still haven't found the right, concise hypothesis and purpose for your research. Secondly, I asked who was the producer of the DNA extraction test. You wrote back that you did it yourself. I suppose you did it yourself, but since you give your own name of the test (DNeary Power Soil Kit 100), you should name the manufacturer. If not, describe what you have modified. And the matter of conclusions. These are still not the conclusions of the research, the lack of specificity and the lack of their practical application.

Author Response

Reviewer 2

Dear Authors, thank you for your answers. Unfortunately, the publication still needs to be corrected.

1. First, I still haven't found the right, concise hypothesis and purpose for your research.

Response: Dear reviewer, according to your suggestion, we have revised the parts of hypothesis and purpose. The revised section is as follow “Various roughage contains different lignin composition, which is closely linked ruminant production. However, few reports discuss the composition of the lignin monomer and its linkage to roughage utilization. Thus, we hypothesized that the lignin monomer composition is a leading marker affecting fiber fractions degradation of roughage. The present study aimed to explore the functional relationship between lignin monomer composition and ruminal fiber fractions degradation in water buffalo, using different roughage sources, which may establish a novel approach to evaluate roughage's nutritional value.”

2. Secondly, I asked who was the producer of the DNA extraction test. You wrote back that you did it yourself. I suppose you did it yourself, but since you give your own name of the test (DNeary Power Soil Kit 100), you should name the manufacturer. If not, describe what you have modified.

Response: Dear reviewer, we have added the manufacturer (QIAGEN) in the paper. And the sentence has been revised as follow “The DNA of microorganisms in rumen content was extracted according to DNeasy PowerSoil Kit 100 manufactured by QIAGEN.”

3. And the matter of conclusions. These are still not the conclusions of the research, the lack of specificity and the lack of their practical application.

Response: Dear reviewer, thanks for your professional suggestion. We have revised the conclusions according to your advice “It was concluded that lignin monomer composition is an important factor affecting fiber fractions degradation in roughage, and the prediction models are as follow: NDFD=0.647-0.071×H/S (R²=0.628, p<0.01), ADFD=0.427-0.045×H/S+0.826×S/G (R²=0.678, p<0.01), ADLD=0.161-1.918×H+3.152×S (R²=0.758, p<0.01), CELD=0.724-0.081×H/S (R²=0.620, p<0.01), HCD=0.522-0.038×H/S+0.649×S/G (R²=0.709, p<0.01). This experiment fills in the blanks of relationship between lignin composition and ruminal fiber fractions degradation. The result provides a new approach for evaluating roughage feedstuffs and improving a better understanding of fiber utilization in ruminants. Moreover, we found that the lignin degradation ability of buffalo is based on its unique rumen microorganisms, but the specific degradation mechanism needs to be further studied.”

Author Response File: Author Response.docx

Reviewer 3 Report

General comments

Objective of the manuscript: The present study aims to investigate the relation between the lignin classes’ coniferyl alcohol (G), ρ-coumaryl alcohol (H), and sinapyl alcohol (S) and ruminal fiber degradability in water buffalo. (Lines 24-26)

Conclusion of the manuscript: The experiment provides a scientific basis for building a new technological method using lignin composition to evaluate buffalo roughage. In addition, this experiment also revealed that buffalo owns rumen microorganisms with lignin degradation potential. (Lines 44-46)

Please read the above points carefully and see if these points are related to each other. All topics in the manuscript must be correlated.

Specific comments

Line (98): Experimental design is: randomized design, Latin square, etc. Choose the correct experimental design and add this information here

Line (170): Describe the full name of COG and KEGG the first time it was mentioned

Table 7: Why did you choose these models? Why does the monomer representing "x" vary between models? Explain this in the manuscript.

Table 8: What is rumen1, rumen2, rumen3 …?. Don't just explain it to me, describe this in the table, maybe as a footnote

Author Response

Reviewer 3

General comments

1. Objective of the manuscript: The present study aims to investigate the relation between the lignin classes’ coniferyl alcohol (G), ρ-coumaryl alcohol (H), and sinapyl alcohol (S) and ruminal fiber degradability in water buffalo. (Lines 24-26)Conclusion of the manuscript: The experiment provides a scientific basis for building a new technological method using lignin composition to evaluate buffalo roughage. In addition, this experiment also revealed that buffalo owns rumen microorganisms with lignin degradation potential. (Lines 44-46)Please read the above points carefully and see if these points are related to each other. All topics in the manuscript must be correlated.

Response: Dear reviewer, thanks for your professional advice. We have considered your suggestion carefully. The abstract has been rewritten, and the revised abstract is as follow “Water buffalo (Bubalus bubalis), known for its unique utilization of low-quality fibrous feeds and out-standing digestion performance, highlighting its role as an animal model in studying fiber fractions degradation. Among roughage, lignin attracted wide attention in ruminant nutrition studies, which affects animal digestibility. Therefore, the present study aims to investigate the functional relation between three lignin monomeric composition of coniferyl alcohol (G), ρ-coumaryl alcohol (H), and sinapyl alcohol (S) and ruminal fiber degradation in water buffalo. Hence, three female water buffaloes (Neri Raffi × Mediterranean, five years old, 480 ± 20 kg) were assigned for an in-vivo study by the nylon-bag method, examining eight kinds of roughage. All the experimental roughage types were analyzed for the effective degradability (ED) of neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), cellulose (CEL) and hemicellulose (HC) fractions. Then, a prediction model for the roughage fiber degradation was established, based on the characteristics of lignin monomer content. The results showed that S, S/G, S/(G+S+H) were positively correlated with the ED of NDF, ADF, CEL and HC; H/S was negatively correlated. For the effective degradability of ADL (ADLD), S, S/(G+S+H) were positively correlated with it; H, H/G, H/S and H/(G+S+H) were negatively correlated. The model with the highest fitting degree was ADLD=0.161-1.918×H+3.152×S (R²=0.758, p<0.01). These results indicated that lignin monomer composition is closely related to the utilization rate of roughage fiber. S-type lignin monomer plays a vital role in the fiber degradation of roughage. The experiment found the effect of lignin monomer composition on the degradation of fiber fractions using buffalo as the experimental animal, and con-structed prediction models providing a scientific basis for building a new technological method using lignin composition to evaluate buffalo roughage. Furthermore, the capacity of ADL degradation of buffalo was proved in this experiment. To further explore the ability of lignin degradation by the buffalo, DNA of rumen microorganisms was extracted for sequencing. The top three composition of rumen mi-croorganisms at genus level were Prevotella_1, 226, Rikenellaceae_RC9_gut_group and Ruminococca-ceae_UCG-011. Six strains with lignin degradation ability were screened from buffalo rumen contents. This experiment also revealed that buffalo owns rumen microorganisms with lignin degradation potential.”

Specific comments

1. Line (98): Experimental design is: randomized design, Latin square, etc. Choose the correct experimental design and add this information here

Response: Dear reviewer, thanks for your professional suggestion, the experimental design is completely randomized design. And we have revised the sentence as follow “Eight types of roughage (rice straw, bracts, maize stalks, peanut vine, sweet potato vine, rice husk, maize silage, vinasse) were used in completely randomized design.”

2. Line (170): Describe the full name of COG and KEGG the first time it was mentioned

Response: Thanks for your suggestion, we have described the full name Cluster of Orthologous Groups of proteins (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) the first time it was mentioned in paper.

3. Table 7: Why did you choose these models? Why does the monomer representing "x" vary between models? Explain this in the manuscript.

Response: Dear reviewer, thanks for your professional suggestion. We have explained the reason why we chose these models in 2.7. Statistical analysis. The revised sentence is as follow “We analyzed the chemical composition and degradation characteristics of samples using a t-test with SPSS 19.0. The correlation between lignin composition and fiber fractions degradation was analyzed with SPSS 19.0. Because of all the correlations are linear, a multiple regression linear model is established. Multiple regression prediction model y = a + b₁x₁ + b₂x₂ + b₃x₃ + ··· + b_mx_m, where y is NDFD, ADFD, CELD, HCD and ADLD, a is the random error constant, b is the independent variable coefficient, x is H content, S content, G content, S / G ratio, H / G ratio, H / S ratio, (G + S + H) content, G / (G + S + H) ratio, S / (G + S + H) ratio and H / (G + S + H) ratio, The regression equation was obtained by stepwise regression of the above model variables with SPSS 19.0. Statistical significance was set at p < 0.05 (significant difference) and p < 0.01 (extremely significant difference).”

The reason why the monomer representing “x” vary between models is added in 3.4. Multiple regression model among lignin composition and fiber fractions degradation. The revised sentence is as follow “The fiber-fractions degradation could be predicted using stepwise regression through the model presented in Table 7. The essence of stepwise regression is to establish an "optimal" multiple linear regression model, so only the monomer representing "x" that have a significant impact on the fiber fractions degradation are retained, and other monomer representing "x" that have no significant impact are excluded. Therefore, the monomer representing "x" in different models are different. The ADL model could explain the degradation (R²) of 0.758, followed by HCD (0.709), ADF (0.678), NDF（0.628）, CELD (0.620). According to these models, the factors affecting fiber fractions degradation could accurately describe and predict the ruminal digestibility of different forages in a similar approach.”

4. Table 8: What is rumen1, rumen2, rumen3 …?. Don't just explain it to me, describe this in the table, maybe as a footnote

Response: Dear reviewer, thanks for your suggestion. We have described in the 2.4. DNA extraction, sequencing of microbial diversity, sequencing data analysis. “To avoid samples disturbance with forage, total six rumen contents samples named rumen0.1, rumen0.2, rumen0.3, rumen0.4, rumen0.5, rumen0.6 were collected after 24 hours fasting of three fistulated buffalo, two samples were collected from each buffalo rumen.” And we have explained as a footnote in Table 8.

Author Response File: Author Response.docx