Effects of Different Additives on the Chemical Composition, Fermentation Profile, In Vitro and In Situ Digestibility of Paper mulberry Silage

Round 1

Reviewer 1 Report

I read the manuscript entitled “Effects of different additives on the chemical composition, fermentation profile, in vitro and in situ digestibility of paper mulberry silage” with great interest.

The silage additives evaluated are pretty common but the novelty of this study is the use of Paper Mulberry as silage, which is a common plant in Asia and its use as silage for ruminant feeding seems to be increasing. So, scientific information on this regard is highly important.

I have a few minor suggestions and concerns as follows:

L39 – replace ‘decreaseng’ by ‘decreasing’

L39 – LAB decrease pH but not ammonia-N. Ammonia-N is less formed when the pH is low. Is not that there was a high amount of ammonia-N and LAB decreased it. Reword it.

L39-40 – Include a reference for this statement or reword it. The word ‘additive’ includes both chemical and biological additives. Use biological additive or inoculant instead of only additive.

L69 – why 45 days?

L85 – Use ‘Van Soest’ instead of ‘Van’.

112 – Remove statistical analysis from this heading

132 – Include the version

143 – Table 1. 90% of DM? ADF greater than NDF (LM)? Please, double check the values presented in Table 1.

225 – Do not initiate a sentence with an abbreviation

228 – There is no proliferation of LAB in the silage. Molasses increase the amount of subtract that will be consumed by LAB, increased lactic acid, which is one of the end-products of the WSC fermentation by LAB. Reword it.

Author Response

I read the manuscript entitled “Effects of different additives on the chemical composition, fermentation profile, in vitro and in situ digestibility of paper mulberry silage” with great interest.

The silage additives evaluated are pretty common but the novelty of this study is the use of Paper Mulberry as silage, which is a common plant in Asia and its use as silage for ruminant feeding seems to be increasing. So, scientific information on this regard is highly important.

I have a few minor suggestions and concerns as follows:

L39 – replace ‘decreaseng’ by ‘decreasing’

Reply: Thanks for your careful review and we have modified it into “decreasing”

L39 – LAB decrease pH but not ammonia-N. Ammonia-N is less formed when the pH is low. Is not that there was a high amount of ammonia-N and LAB decreased it. Reword it.

Reply: It have been revised into: Lactic acid bacteria (LAB) are capable of prompting the production of lactic acid then decreasing pH and inhibiting the production of ammonia-N content of silage

L39-40 – Include a reference for this statement or reword it. The word ‘additive’ includes both chemical and biological additives. Use biological additive or inoculant instead of only additive.

Reply: It have been changed into “biological additive” and added a reference which deeply review the lactic acid bacteria as a biological additive in silage production.

Muck, R. E., E. M. G. Nadeau, T. A. McAllister, F. E. Contreras-Govea, M. C. Santos, and L. Kung, Jr. 2018. Silage review: Recent advances and future uses of silage additives. J Dairy Sci 101(5):3980-4000.

L69 – why 45 days?

Reply: normal, the ensiling more than 30 days will be in a stable status. So some studys is 30 days, 35 days, and 56 days. Please see the following reference. We chose 45 days, because 45 days is enough for paper mulberry to ensiling and considering the real situation of our lab work arrangement.

1. 30 days: The reconstitution mechanism of napier grass microiota during the ensiling of alfalfa and their contributions to fermentation quality of silage. 2020, Bioresource Technology.
2. 35 days: Additives affect the distribution of metabolic profile, microbial communities and antibiotic resistance genes in high-moisture sweet corn kernel silage. 2020, Bioresource Technology.
3. 56 days: Microbial community dynamics during alfalfa silage with or without clostridial fermentation. 2020, Scientific Report.

L85 – Use ‘Van Soest’ instead of ‘Van’.

Reply: thanks for your kindly advise and we have changed it to “Van Soest”

112 – Remove statistical analysis from this heading

Reply: Thanks for your carefully review and we have deleted it.

132 – Include the version

Reply: we have added the version, which is version 9.0

143 – Table 1. 90% of DM? ADF greater than NDF (LM)? Please, double check the values presented in Table 1.

Reply: We have rechecked our data and found we before using air-dried basis to calculate the DM content, it is not common use in silage research, and we have revised it into as-fed basis. The type wrong is also occurred for the NDF of LM group should be 41.39, not 31.39 (Table 1).

225 – Do not initiate a sentence with an abbreviation

Reply: we have reworded the sentence as: The crucial factor for successful silage fermentation is WSC. To avoid the abbreviation in the beginning of a sentence.

228 – There is no proliferation of LAB in the silage. Molasses increase the amount of subtract that will be consumed by LAB, increased lactic acid, which is one of the end-products of the WSC fermentation by LAB. Reword it.

Reply: Thanks for your revision, and we have reworded it to: “Molasses increase the amount of substrate that will be consumed by LAB, and in-creased lactic acid, which is one of the end-products of the WSC fermentation by LAB, and the lactic acid can lower the silage pH value more quickly than without its addition”

Reviewer 2 Report

Abstract
The description of the methodology presented the summary is quite unclear.
The conclusion seems vague, stating that LAB, MOL and, especially, the combination of both can improve fermentation and digestibility.

Introduction
The introduction is too synthesized, leaving room to further explore this section. There should be an explanation of the importance of the pH of silages and more information on the plant used.
The objective described in the introduction is slightly different from what is in the abstract.

Material and Methods
The indication of the number of replicates must come right at the beginning of the materials and methods section.
The rumen came from fistulated cows. However, the article does not refer to any animal welfare authorization for the use of these animals in the test.
The chemical composition of the animals' diet must be indicated.

Results
It is not possible to understand what table 3 refers to. The pH value is too high for silages, always above 4, although there is a significant reduction in pH when comparing the control group with the treatment groups. In table 3, there are doubts about the significant differences verified in the pH of NH3, because with the SE values, the differences in the averages are practically null. There is great variability of values, this said, the coefficient of variation should be presented, or the maximum and minimum values. In gas production, as a rule, the GP value used is 24h and not 48h, as used in this study.
Discussion
The differences between Table 2 and 3 are not noticeable.
The discussion, despite being divided by themes, is not clear. Conclusion

Author Response

Reviewer2

Abstract

The description of the methodology presented the summary is quite unclear.

The conclusion seems vague, stating that LAB, MOL and, especially, the combination of both can improve fermentation and digestibility.

Reply: Thanks for your kindly suggestions, we have made our methodology more specific in the abstract. Added the concentration of additives and the ensiling temperatures, which made the silage process more specific. “Four groups consist of PM silage without any additive as the control group (CON), CON with either 5 ×10⁶ CFU per gram of fresh PM weight of lactic acid bacteria (Lactobacillus plantarum) (LAB) or 3% fresh PM weight of molasses (MOL) added to the PM silage, respectively, or both LAB and MOL were added (LM). After 45 days of ensiling at 20 °C…..”

And we also reword the in situ result and our conclusions, which give more specific results of additives on the PM silage femmentation and digestibility, which are as folowwing: “ Results of in situ experiment suggested that effective digestibility of PM silage dry matter as well as acid detergent fiber digestibility was higher in LM group than the CON group (P < 0.05). In conclusion, LAB, MOL, and their combination can improve the PM silage fermentation, didn’t affect PM silage in vitro digestibility, and improve the in situ digestibility of dry matter and acid detergent fiber.”

Introduction

The introduction is too synthesized, leaving room to further explore this section. There should be an explanation of the importance of the pH of silages and more information on the plant used.

The objective described in the introduction is slightly different from what is in the abstract.

Reply: Thanks for your kindly suggestions and comments. We agree that there is still need some improve for our introduction parts. We mainly made the three aspects of revision as following:

We expanded the PM usage in the introduction parts, besides in animal production industry, we also give a brief introduction of PM usage in other aspects. Because of its strong rooting ability and rapid growth, PM is considered a plant capable of providing environmental afforestation in many areas [29]. Two decades ago, wide areas of PM were planted to perform the soil erosion prevention function in China [30].

We strong agree with you that more detailed information should be included in the pH related and highlight its importance for silage fermentation. So we added the following information. “pH is one of the most important indices in the silage fermentation, low pH can inhibit the bacteria activity and avoid the nutrients be fermented and loses [28]. Lactic acid is one of the most important acids, which is widely studied and play the most important role in downregulating the silage pH [28]. We must be pay attention to these two indices when evaluating the silage fermentation quality.”

We have revised the objective results as following: In this study, LAB, MOL, and their combination were selected as silage additives. We aimed to explore the effects of these different silage additives on the chemical composition, fermentation profile, as well as in vitro and in situ digestibility of PM silage.

Material and Methods

The indication of the number of replicates must come right at the beginning of the materials and methods section.

The rumen came from fistulated cows. However, the article does not refer to any animal welfare authorization for the use of these animals in the test.

The chemical composition of the animals' diet must be indicated.

Reply: we have double checked our replicates again and also included the animal welfare authorization and diet composition (added in the supplement materials part) in the M&M parts, please check it in the revised version.

All the animal use procedures carried out during this experimental period were approved by China Agriculture University Laboratory Animal Welfare and Animal Experimental Ethical Faculty (protocol number: AW81102202-1-2). All the cows were in a stage of high lactation, having a diet of 60% forage and 40% concentrate with net energy of 8.16 MJ/kg, the ingredients and chemical composition of cows’ diet in Table S1.

Results

It is not possible to understand what table 3 refers to. The pH value is too high for silages, always above 4, although there is a significant reduction in pH when comparing the control group with the treatment groups. In table 3, there are doubts about the significant differences verified in the pH of NH3, because with the SE values, the differences in the averages are practically null. There is great variability of values, this said, the coefficient of variation should be presented, or the maximum and minimum values.

Reply: Thanks for your comments and we have double checked all the data and there is something wrong for the result and we have re-statistics all the data and revised them marked in yellow and the results and discussion also reworded based on the revised result. Please check the revised version.

In gas production, as a rule, the GP value used is 24h and not 48h, as used in this study.

Reply: Thanks for your suggestions and comments. We agree that 24 h is a most common use for in vitro fermentation. But recently, we realized there is still producing of gas between the 24 to 48h and we also reference some published paper, which also using the 48h, so we decided to use the 48 h GP as our final indices. The following papers are using the 48 h GP.

Effects of Different Parts on the Chemical Composition, Silage Fermentation Profile, In Vitro and In Situ Digestibility of Paper Mulberry.

In vitro and in vivo Studies of Soybean Peptides on Milk Production, Rumen Fermentation, Ruminal Bacterial Community, and Blood Parameters in Lactating Dairy Cows

Discussion

The differences between Table 2 and 3 are not noticeable.

The discussion, despite being divided by themes, is not clear. Conclusion

Reply: we have thoroughly revised our results, discussion, and conclusion parts, please check the revised version.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

The authors should explain better what the values in table 3 represent, I think they refer to the parameters of the rumen fluid, but this is not written anywhere.

Author Response

Thanks for your careful review of our manuscript again. We strongly agree with you that the description of VFA et al. in table 3 seems vague. We have carefully gone through our manuscript and revised related problems in light blue.

We have revised the table 3 and table 4 titles as follows, which will be easier for readers to know what the parameters refer to.

Table 3. Effects of paper mulberry silage treated with different additives on the in vitro degradability of DM, ruminal liquid pH, VFAs, and ammonia after 48 h of incubation

Table 4. Effects of paper mulberry silage treated with different additives on the in vitro gas production, and kinetic parameters after 48 h of incubation

And also, we double check the results and discussion parts again. We revised the result parts as follows:

The in vitro experiment results are shown in Tables 3 and 4. No significant difference in PM silage IVDMD was found among all the groups. The in vitro ruminal fluid NH₃-N content of the LM group was higher than the CON and MOL groups, however, the ruminal fluid pH in the LM group was lower than that in CON and MOL groups (P < 0.05). In vitro ruminal fluid acetic acid, propionic acid, isobutyric acid, valeric acid, isovaleric acid, and total VFA also occurred at similar levels among all four groups. By contrast, the ruminal fluid butyric acid in the CON group was at a lower level than the LAC and LM group (P < 0.05).

Thanks for your review again and we appreciate your work to help us improve our manuscript continuously.

Author Response File: Author Response.docx