Replacing Alfalfa with Paper Mulberry in Total Mixed Ration Silages: Effects on Ensiling Characteristics, Protein Degradation, and In Vitro Digestibility
Round 1
Reviewer 1 Report
The manuscript was largely improved in the language and content exposition. Statistics is now well presented and very clear. Also, the tables were corrected and simplified.
I really appreciate the author's revisions and corrections.
I only suggest revising the form of the manuscript, if it is alike the journal given form, especially for tables, text size, and spatial distribution.
Author Response
Point 1: The manuscript was largely improved in the language and content exposition. Statistics is now well presented and very clear. Also, the tables were corrected and simplified. I really appreciate the author's revisions and corrections.
Response 1: Thank you very much for the comments.
Point 2: I only suggest revising the form of the manuscript, if it is alike the journal given form, especially for tables, text size, and spatial distribution.
Response 2: We appreciate the valuable suggestions. We checked form of manuscript according to the journal given form. The line spacing of manuscript title was corrected to 12 pounds. Please see [Page#1; Lines 2 to 4]. “37°C” was corrected to “37 °C” and “30°C” was corrected to “30 °C”. Please see [Page#3; Lines 121 and Line 124]. The title of table 4 was moved to next page. Please see [Page#7; Line 235]. The title of table 7 was moved to next page. Please see [Page#9; Line 286]. In results section, we added the point to 3.7 (in vitro digestibility of 56-day TMR silages) section. Please see [Page#9; Line 293]. In author contributions and reference section, short front spacing was corrected to 12 pounds. Please see [Page#12; Line 450] and [Page#13; Line 472].
Author Response File: 
Author Response.docx
Reviewer 2 Report
Improved work compared to the initial version, but still with some suggested changes that have not been modified.
Global comments:
- Please it would be interesting to make an index of abbreviations, it would serve to better understand the text.
- What about amino acids? It would be interesting to add this information both in the introduction and above all in the discussion, since the CP is not important but the AA. If the analysis has not been carried out in the laboratory, the ideal would be even to do it with theoretical data.
Specific comments:
The data was not checked for normality? kurtosis? Can anova be applied? If it has been done it must be specified, if it has not been done it should be done.
(Table 1) No standard error? This should wear.
¿Do you think that the number of samples is representative?
Author Response
Comments and Suggestions for Authors (Global comments)
Point 1: Improved work compared to the initial version, but still with some suggested changes that have not been modified.
Response 1: Thanks for the comments.
Point 2: Please it would be interesting to make an index of abbreviations, it would serve to better understand the text.
Response 2: We appreciate the valuable suggestion. Acronym index table was added to better understand the manuscript. Please see [Page#13; Lines 471].
Point 3: What about amino acids? It would be interesting to add this information both in the introduction and above all in the discussion, since the CP is not important but the AA. If the analysis has not been carried out in the laboratory, the ideal would be even to do it with theoretical data.
Response 3: We appreciate the valuable suggestion. Some information was added to the manuscript. Please see [Page#11; Lines 384 to 389].
Amino acid nutrition of the diet can influence animal performance, and balanced amino acid nutrition of the diet is essential for maximizing nutrient utilization and productivity of dairy and beef cattle. Quantitative evaluation of amino acid composition of the diet offers important reference to meeting the nutritional needs of dairy cows.
The aim of this study was preliminary evaluation on the dynamics of fermentation characteristics, chemical composition, protein degradation and in vitro digestibility of total mixed ration (TMR) silages when paper mulberry (Broussonetia papyrifera L., RY) was substituted for alfalfa at different ratios. Our results showed that RY substitution had no adverse effect on fermentation quality and nutritional composition of TMR silages. After ensiling, the contents of non-protein nitrogen, peptide nitrogen and free amino acid nitrogen for RY18 silages was lower than that of the control, but the buffer soluble protein and acid detergent soluble protein fractions was not different among the all silages. Digestibility of dry matter and crude protein for RY27 and RY36 silages was lower than the control, but there was no difference between control and RY18 silages. Therefore, it appears that TMR silages produced with RY to alfalfa ratio of 18:18 was suitable as a result of balance the silage quality, protein degradation and in vitro digestibility.
Amino acid levels (including essential amino acid and non-essential amino acid) are not determined but the total free amino acid nitrogen in this experiment. Further quantitative evaluation of amino acid degradation in TMR silages is essential to add the more information on accurate animal feeding and production when RY is introduced. Thanks for pointing out the direction of our future research. Buddle of thanks for the valuable suggestion.
Specific comments:
Point 4: The data was not checked for normality? kurtosis? Can ANOVA be applied? If it has been done it must be specified, if it has not been done it should be done.
Response 4: Thanks for the comments. The sentence “Prior to statistical analysis, all data is subjected to Homogeneity test of variance. The normality was tested with Shapiro–Wilk test. All data can be analyzed by ANOVA” was added to statistical analysis section. Please see [Page#4; Lines 168 to 170]. The analytical results of were as follows:
|
Table 1. Homogeneity test of variance of the chemical composition and in vitro digestibility. |
||||
|
Items |
levene statistics |
df1 |
df2 |
sig. |
|
Fresh materials |
|
|
|
|
|
Dry matter |
0.902 |
4 |
10 |
0.499 |
|
Crude protein |
1.378 |
4 |
10 |
0.309 |
|
Neutral detergent fiber |
0.312 |
4 |
10 |
0.864 |
|
Acid detergent fiber |
3.074 |
4 |
10 |
0.074 |
|
Acid detergent lignin |
2.132 |
4 |
10 |
0.151 |
|
Water soluble carbohydrates |
0.413 |
4 |
10 |
0.796 |
|
Buffer capacity |
3.245 |
4 |
10 |
0.066 |
|
Condensed tannin |
0.616 |
4 |
10 |
0.661 |
|
Hydrolysable tannin |
0.604 |
4 |
10 |
0.668 |
|
After ensiling |
|
|
|
|
|
Dry matter |
2.302 |
4 |
10 |
0.130 |
|
Crude protein |
1.801 |
4 |
10 |
0.205 |
|
Neutral detergent fiber |
0.148 |
4 |
10 |
0.960 |
|
Acid detergent fiber |
0.919 |
4 |
10 |
0.490 |
|
Acid detergent lignin |
2.322 |
4 |
10 |
0.156 |
|
Water soluble carbohydrates |
2.328 |
4 |
10 |
0.127 |
|
Buffer capacity |
0.965 |
4 |
10 |
0.468 |
|
Condensed tannin |
1.714 |
4 |
10 |
0.223 |
|
Hydrolysable tannin |
0.749 |
4 |
10 |
0.580 |
|
In vitro dry matter digestibility |
1.808 |
4 |
10 |
0.204 |
|
In vitro crude protein digestibility |
0.984 |
4 |
10 |
0.459 |
|
In vitro neural detergent fiber digestibility |
1.526 |
4 |
10 |
0.267 |
The significance value of chemical composition (including fresh materials and ensiling after 56 days) and in vitro digestibility (ensiling after 56 days) parameters was more than 0.05 (Table 1). Therefore, this result is consistent with the homogeneity test of variance.
|
Table 2. Normality test of the chemical composition and in vitro digestibility. |
||||||
|
Items |
Kolmogorov-Smirnov |
Shapiro-Wilk |
||||
|
statistics |
df |
sig. |
statistics |
df |
sig. |
|
|
Fresh materials |
|
|
|
|
|
|
|
Dry matter |
0.144 |
15 |
0.200 |
0.937 |
15 |
0.345 |
|
Crude protein |
0.129 |
15 |
0.200 |
0.955 |
15 |
0.611 |
|
Neutral detergent fiber |
0.088 |
15 |
0.200 |
0.973 |
15 |
0.896 |
|
Acid detergent fiber |
0.142 |
15 |
0.200 |
0.947 |
15 |
0.486 |
|
Acid detergent lignin |
0.107 |
15 |
0.200 |
0.975 |
15 |
0.925 |
|
Water soluble carbohydrates |
0.140 |
15 |
0.200 |
0.969 |
15 |
0.836 |
|
Buffer capacity |
0.174 |
15 |
0.200 |
0.917 |
15 |
0.172 |
|
Condensed tannin |
0.155 |
15 |
0.200 |
0.915 |
15 |
0.161 |
|
Hydrolysable tannin |
0.171 |
15 |
0.200 |
0.912 |
15 |
0.146 |
|
After ensiling |
|
|
|
|
|
|
|
Dry matter |
0.112 |
15 |
0.200 |
0.959 |
15 |
0.670 |
|
Crude protein |
0.152 |
15 |
0.200 |
0.938 |
15 |
0.359 |
|
Neutral detergent fiber |
0.142 |
15 |
0.200 |
0.971 |
15 |
0.878 |
|
Acid detergent fiber |
0.167 |
15 |
0.200 |
0.933 |
15 |
0.303 |
|
Acid detergent lignin |
0.176 |
15 |
0.200 |
0.962 |
15 |
0.719 |
|
Water soluble carbohydrates |
0.180 |
15 |
0.200 |
0.949 |
15 |
0.507 |
|
Buffer capacity |
0.114 |
15 |
0.200 |
0.977 |
15 |
0.949 |
|
Condensed tannin |
0.169 |
15 |
0.200 |
0.911 |
15 |
0.141 |
|
Hydrolysable tannin |
0.141 |
15 |
0.200 |
0.912 |
15 |
0.145 |
|
In vitro dry matter digestibility |
0.131 |
15 |
0.200 |
0.974 |
15 |
0.908 |
|
In vitro crude protein digestibility |
0.144 |
15 |
0.200 |
0.946 |
15 |
0.468 |
|
In vitro neural detergent fiber digestibility |
0.177 |
15 |
0.200 |
0.927 |
15 |
0.245 |
The significance value of chemical composition (including fresh materials and ensiling after 56 days) and in vitro digestibility (ensiling after 56 days) parameters was more than 0.05 according to Shapiro-Wilk test (Table 2). Therefore, this result is consistent with normality test.
|
Table 3. Homogeneity test of variance of the fermentation quality, protein and microbial composition. |
||||
|
Items |
levene statistics |
df1 |
df2 |
sig. |
|
0 day |
|
|
|
|
|
Non-protein nitrogen |
1.213 |
4 |
10 |
0.365 |
|
Peptide nitrogen |
1.314 |
4 |
10 |
0.329 |
|
Free amino acid nitrogen |
0.129 |
4 |
10 |
0.968 |
|
Ammonia nitrogen |
2.049 |
4 |
10 |
0.163 |
|
Neutral detergent soluble protein |
1.009 |
4 |
10 |
0.448 |
|
Acid detergent soluble protein |
0.811 |
4 |
10 |
0.546 |
|
Acid detergent insoluble protein |
3.052 |
4 |
10 |
0.069 |
|
7 day |
|
|
|
|
|
pH |
1.467 |
4 |
10 |
0.283 |
|
Latic acid |
1.027 |
4 |
10 |
0.171 |
|
Acetic acid |
2.203 |
4 |
10 |
0.142 |
|
Propionic acid |
1.081 |
4 |
10 |
0.516 |
|
Non-protein nitrogen |
0.650 |
4 |
10 |
0.640 |
|
Peptide nitrogen |
2.473 |
4 |
10 |
0.112 |
|
Free amino acid nitrogen |
1.012 |
4 |
10 |
0.446 |
|
Ammonia nitrogen |
1.121 |
4 |
10 |
0.400 |
|
Neutral detergent soluble protein |
2.218 |
4 |
10 |
0.140 |
|
Acid detergent soluble protein |
0.499 |
4 |
10 |
0.737 |
|
Acid detergent insoluble protein |
1.888 |
4 |
10 |
0.189 |
|
Lactic acid bacteria |
1.466 |
4 |
10 |
0.168 |
|
Aerobic bacteria |
1.059 |
4 |
10 |
0.144 |
|
14 day |
|
|
|
|
|
pH |
1.412 |
4 |
10 |
0.299 |
|
Latic acid |
1.725 |
4 |
10 |
0.221 |
|
Acetic acid |
1.204 |
4 |
10 |
0.368 |
|
Propionic acid |
0.355 |
4 |
10 |
0.835 |
|
Non-protein nitrogen |
0.981 |
4 |
10 |
0.460 |
|
Peptide nitrogen |
1.577 |
4 |
10 |
0.254 |
|
Free amino acid nitrogen |
1.942 |
4 |
10 |
0.136 |
|
Ammonia nitrogen |
3.076 |
4 |
10 |
0.055 |
|
Neutral detergent soluble protein |
0.469 |
4 |
10 |
0.873 |
|
Acid detergent soluble protein |
0.423 |
4 |
10 |
0.789 |
|
Acid detergent insoluble protein |
1.150 |
4 |
10 |
0.388 |
|
Lactic acid bacteria |
1.759 |
4 |
10 |
0.211 |
|
Aerobic bacteria |
1.013 |
4 |
10 |
0.279 |
|
28 day |
|
|
|
|
|
pH |
2.794 |
4 |
10 |
0.085 |
|
Latic acid |
1.129 |
4 |
10 |
0.397 |
|
Acetic acid |
2.013 |
4 |
10 |
0.169 |
|
Propionic acid |
0.225 |
4 |
10 |
0.918 |
|
Non-protein nitrogen |
0.761 |
4 |
10 |
0.574 |
|
Peptide nitrogen |
2.088 |
4 |
10 |
0.157 |
|
Free amino acid nitrogen |
1.116 |
4 |
10 |
0.172 |
|
Ammonia nitrogen |
2.000 |
4 |
10 |
0.171 |
|
Neutral detergent soluble protein |
2.141 |
4 |
10 |
0.116 |
|
Acid detergent soluble protein |
1.397 |
4 |
10 |
0.303 |
|
Acid detergent insoluble protein |
1.083 |
4 |
10 |
0.415 |
|
Lactic acid bacteria |
0.079 |
4 |
10 |
0.277 |
|
Aerobic bacteria |
1.012 |
4 |
10 |
0.204 |
|
56 day |
|
|
|
|
|
pH |
2.933 |
4 |
10 |
0.076 |
|
Latic acid |
0.056 |
4 |
10 |
0.993 |
|
Acetic acid |
3.291 |
4 |
10 |
0.058 |
|
Propionic acid |
0.974 |
4 |
10 |
0.463 |
|
Non-protein nitrogen |
0.673 |
4 |
10 |
0.626 |
|
Peptide nitrogen |
1.651 |
4 |
10 |
0.237 |
|
Free amino acid nitrogen |
1.467 |
4 |
10 |
0.283 |
|
Ammonia nitrogen |
1.910 |
4 |
10 |
0.185 |
|
Neutral detergent soluble protein |
1.185 |
4 |
10 |
0.375 |
|
Acid detergent soluble protein |
1.094 |
4 |
10 |
0.411 |
|
Acid detergent insoluble protein |
1.494 |
4 |
10 |
0.276 |
|
Lactic acid bacteria |
1.741 |
4 |
10 |
0.169 |
|
Aerobic bacteria |
1.218 |
4 |
10 |
0.174 |
The significance value of fermentation quality, protein and microbial composition during ensiling was more than 0.05 (Table 3). Therefore, this result is consistent with the homogeneity test of variance.
|
Table 4. Normality test of the fermentation quality, protein and microbial composition. |
||||||
|
Items |
Kolmogorov-Smirnov |
Shapiro-Wilk |
||||
|
statistics |
df |
sig. |
statistics |
df |
sig. |
|
|
0 day |
|
|
|
|
|
|
|
Non-protein nitrogen |
0.165 |
15 |
0.200 |
0.924 |
15 |
0.225 |
|
Peptide nitrogen |
0.181 |
15 |
0.197 |
0.910 |
15 |
0.136 |
|
Free amino acid nitrogen |
0.104 |
15 |
0.200 |
0.979 |
15 |
0.963 |
|
Ammonia nitrogen |
0.175 |
15 |
0.200 |
0.925 |
15 |
0.233 |
|
Neutral detergent soluble protein |
0.177 |
15 |
0.200 |
0.943 |
15 |
0.420 |
|
Acid detergent soluble protein |
0.107 |
15 |
0.200 |
0.989 |
15 |
0.999 |
|
Acid detergent insoluble protein |
0.174 |
15 |
0.200 |
0.930 |
15 |
0.274 |
|
7 day |
|
|
|
|
|
|
|
pH |
0.179 |
15 |
0.200 |
0.918 |
15 |
0.181 |
|
Latic acid |
0.178 |
15 |
0.200 |
0.949 |
15 |
0.507 |
|
Acetic acid |
0.169 |
15 |
0.200 |
0.904 |
15 |
0.110 |
|
Propionic acid |
0.164 |
15 |
0.200 |
0.936 |
15 |
0.337 |
|
Non-protein nitrogen |
0.205 |
15 |
0.089 |
0.884 |
15 |
0.054 |
|
Peptide nitrogen |
0.169 |
15 |
0.200 |
0.876 |
15 |
0.041 |
|
Free amino acid nitrogen |
0.104 |
15 |
0.200 |
0.938 |
15 |
0.356 |
|
Ammonia nitrogen |
0.231 |
15 |
0.030 |
0.890 |
15 |
0.068 |
|
Neutral detergent soluble protein |
0.167 |
15 |
0.200 |
0.942 |
15 |
0.413 |
|
Acid detergent soluble protein |
0.134 |
15 |
0.200 |
0.936 |
15 |
0.337 |
|
Acid detergent insoluble protein |
0.203 |
15 |
0.096 |
0.902 |
15 |
0.101 |
|
Lactic acid bacteria |
0.153 |
15 |
0.200 |
0.907 |
15 |
0.216 |
|
Aerobic bacteria |
0.107 |
15 |
0.200 |
0.911 |
15 |
0.458 |
|
14 day |
|
|
|
|
|
|
|
pH |
0.207 |
15 |
0.083 |
0.930 |
15 |
0.272 |
|
Latic acid |
0.096 |
15 |
0.200 |
0.965 |
15 |
0.778 |
|
Acetic acid |
0.149 |
15 |
0.200 |
0.904 |
15 |
0.111 |
|
Propionic acid |
0.138 |
15 |
0.200 |
0.935 |
15 |
0.319 |
|
Non-protein nitrogen |
0.217 |
15 |
0.056 |
0.898 |
15 |
0.062 |
|
Peptide nitrogen |
0.162 |
15 |
0.200 |
0.936 |
15 |
0.340 |
|
Free amino acid nitrogen |
0.189 |
15 |
0.200 |
0.969 |
15 |
0.373 |
|
Ammonia nitrogen |
0.153 |
15 |
0.200 |
0.939 |
15 |
0.371 |
|
Neutral detergent soluble protein |
0.168 |
15 |
0.200 |
0.930 |
15 |
0.275 |
|
Acid detergent soluble protein |
0.176 |
15 |
0.200 |
0.931 |
15 |
0.280 |
|
Acid detergent insoluble protein |
0.138 |
15 |
0.200 |
0.939 |
15 |
0.373 |
|
Lactic acid bacteria |
0.203 |
15 |
0.200 |
0.954 |
15 |
0.501 |
|
Aerobic bacteria |
0.141 |
15 |
0.200 |
0.918 |
15 |
0.419 |
|
28 day |
|
|
|
|
|
|
|
pH |
0.165 |
15 |
0.200 |
0.970 |
15 |
0.865 |
|
Latic acid |
0.142 |
15 |
0.200 |
0.959 |
15 |
0.679 |
|
Acetic acid |
0.142 |
15 |
0.200 |
0.925 |
15 |
0.228 |
|
Propionic acid |
0.152 |
15 |
0.200 |
0.963 |
15 |
0.741 |
|
Non-protein nitrogen |
0.123 |
15 |
0.200 |
0.949 |
15 |
0.511 |
|
Peptide nitrogen |
0.144 |
15 |
0.200 |
0.957 |
15 |
0.640 |
|
Free amino acid nitrogen |
0.250 |
15 |
0.200 |
0.947 |
15 |
0.616 |
|
Ammonia nitrogen |
0.167 |
15 |
0.200 |
0.949 |
15 |
0.507 |
|
Neutral detergent soluble protein |
0.162 |
15 |
0.200 |
0.926 |
15 |
0.235 |
|
Acid detergent soluble protein |
0.136 |
15 |
0.200 |
0.952 |
15 |
0.551 |
|
Acid detergent insoluble protein |
0.105 |
15 |
0.200 |
0.942 |
15 |
0.413 |
|
Lactic acid bacteria |
0.146 |
15 |
0.200 |
0.962 |
15 |
0.519 |
|
Aerobic bacteria |
0.139 |
15 |
0.200 |
0.927 |
15 |
0.647 |
|
56 day |
|
|
|
|
|
|
|
pH |
0.207 |
15 |
0.082 |
0.908 |
15 |
0.125 |
|
Latic acid |
0.148 |
15 |
0.200 |
0.944 |
15 |
0.442 |
|
Acetic acid |
0.132 |
15 |
0.200 |
0.910 |
15 |
0.138 |
|
Propionic acid |
0.174 |
15 |
0.200 |
0.925 |
15 |
0.232 |
|
Non-protein nitrogen |
0.103 |
15 |
0.200 |
0.976 |
15 |
0.938 |
|
Peptide nitrogen |
0.159 |
15 |
0.200 |
0.965 |
15 |
0.779 |
|
Free amino acid nitrogen |
0.160 |
15 |
0.200 |
0.944 |
15 |
0.432 |
|
Ammonia nitrogen |
0.211 |
15 |
0.071 |
0.889 |
15 |
0.057 |
|
Neutral detergent soluble protein |
0.132 |
15 |
0.200 |
0.951 |
15 |
0.542 |
|
Acid detergent soluble protein |
0.124 |
15 |
0.200 |
0.973 |
15 |
0.905 |
|
Acid detergent insoluble protein |
0.170 |
15 |
0.200 |
0.945 |
15 |
0.445 |
|
Lactic acid bacteria |
0.181 |
15 |
0.200 |
0.933 |
15 |
0.471 |
|
Aerobic bacteria |
0.142 |
15 |
0.200 |
0.915 |
15 |
0.508 |
The significance value of the fermentation quality, protein and microbial composition during ensiling was more than 0.05 (Table 4). Therefore, this result is consistent with normality test.
Point 5: (Table 1) No standard error? This should wear.
Response 5: Thanks for point out. Standard error of means was added to the table 1. Please see [Page#5; Lines 194 and 197].
Point 6: Do you think that the number of samples is representative?
Response 6: Thanks for the comments. In this study, TMR were made with alfalfa and RY mixtures (36.0%), maize meal (35.0%), oat grass (10.0%), soybean meal (7.5%), brewers’ grain (5.0%), wheat bran (5.0%), pre-mix (1.0%), and salt (0.5%) on dry matter basis, respectively. The alfalfa and RY mixtures were made in the following ratios of dry matter: 36:0 (RY0), 27:9 (RY9), 18:18 (RY18), 9:27 (RY27) and 0:36 (RY36). The experimental diets were formulated according to Chinese Feeding Standard for Dairy Cattle [1]. Five storage periods (0 d, 7 d, 14 d, 28 d and 56 d) were choose basing on the critical period for silage. For each treatment, three replicates of silage samples were prepared and a total of 75 mini silos were obtained for this experiment. After 0, 7, 14, 28 and 56 days of ensiling respectively, triplicate samples of silage from each treatment were opened. Numerous studies [2-5] also set three triplicates of each treatment to evaluate the feed value for silage.
According to previous studies [6-7], a total of one hundred and thirty-five filter bags (five treatments × three individual samples × three filter bags per sample × three runs) were prepared for in vitro digestibility analysis in this study, thus each treatment contained nine replicates.
Reference
[1] Association JGFFS. Guide book for quality evaluation of forage. Tokyo, Japan. 1994, 82-87.
[2] Gao R, Luo Y, Xu S, Wang M, Sun Z, Wang L, Yu, Z. Effects of replacing ensiled-alfalfa with fresh-alfalfa on dynamic fermentation characteristics, chemical compositions, and protein fractions in fermented total mixed ration with different additives. Animals, 2021, 11, 572.
[3] Xie Y, Xu S, Li W, Bao J, Jia T, Yu Z. Effects of the application of Lactobacillus plantarum Inoculant and potassium sorbate on the fermentation quality, in vitro digestibility and aerobic stability of total mixed ration silage based on alfalfa silage. Animals, 2020, 12, 2229.
[4] Du Z, Sun L, Chen C, Lin J, Yang F, Cai Y. Exploring microbial community structure and metabolic gene clusters during silage fermentation of paper mulberry, a high-protein woody plant. Animal Feed Science and Technology, 2021, 275, 114766.
[5] Tian P, Niu D, Zuo S, Jiang D, Li R, Xu C. Vitamin A and E in the total mixed ration as influenced by ensiling and the type of herbage. Science of The Total Environment, 2020, 746, 141239.
[6] Guo L, Yao D, Li D, Lin Y, Bureenok S, Ni K, Yang F. Effects of lactic acid bacteria isolated from rumen fluid and feces of dairy cows on fermentation quality, microbial community, and in vitro digestibility of alfalfa silage. Frontiers in microbiology, 2020, 10, 2998.
[7] Lei C, Dong Z, Li J, Shao T. Ensiling characteristics, in vitro rumen fermentation, microbial communities and aerobic stability of low‐dry matter silages produced with sweet sorghum and alfalfa mixtures. Journal of the Science of Food and Agriculture, 2019, 99, 2140-2151.
[5] Tian P, Niu D, Zuo S, Jiang D, Li R, Xu C. Vitamin A and E in the total mixed ration as influenced by ensiling and the type of herbage. Science of The Total Environment, 2020, 746, 141239.
[6] Guo L, Yao D, Li D, Lin Y, Bureenok S, Ni K, Yang F. Effects of lactic acid bacteria isolated from rumen fluid and feces of dairy cows on fermentation quality, microbial community, and in vitro digestibility of alfalfa silage. Frontiers in microbiology, 2020, 10, 2998.
[7] Lei C, Dong Z, Li J, Shao T. Ensiling characteristics, in vitro rumen fermentation, microbial communities and aerobic stability of low‐dry matter silages produced with sweet sorghum and alfalfa mixtures. Journal of the Science of Food and Agriculture, 2019, 9
Author Response File: Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The research presented cover a useful topic
It provide the ground information to continue in vivo evaluation of novel feeds
The research seems to be adequately performed however there numerous details missing in the M&M section which need to be clarify to confirm that the experimental design and statistical analysis are valid.
Methodological procedure details are missing for some variables
Otherwise the discussion seems appropriated and only need to moderate the conclusion.
(see attached file)
However, given the missing methodological details my recommendation is conservative until further details are provided by authors in a revised version.
Comments for author File: 
Comments.pdf
Author Response
Dear reviewer:
We corrected the manuscript carefully according to the comments. Please see the attachment.
Author Response File: Author Response.docx
Reviewer 2 Report
Dear Authors,
this manuscript is aimed at an interesting purpose, with valuable results. However, it needs to be written again. The references, especially in the introduction, are not responding to the sentences. Acronyms are chaotic and they do not be the same as the tables. Statistic analysis needs references and model formulas with variable factors as reported in the tables. The chemical analysis of the forage pre-fermentation is reported without statistics, nevertheless, is commented as different. All the experimental design of the in vitro trial is missing. No mention of numbers of reactors per treatment and about fermentation conditions.
Author Response
Dear editor and reviewer,
We revised the manuscript carefully basing on the comments, please see the attachment. We hope that revised manuscript will be good quality than before.
Kind regards,
Rongrong Li
Author Response File: Author Response.docx
Reviewer 3 Report
Global comments:
- Please it would be interesting to make an index of abbreviations, it would serve to better understand the text.
- General comment about tables: The tables are in different pages, please wathc the format. For example punctuation.
- What about amino acids? It would be interesting to add this information both in the introduction and above all in the discussion, since the CP is not important but the AA. If the analysis has not been carried out in the laboratory, the ideal would be even to do it with theoretical data.
- I think a more in-depth study should be done on the nutrients that cause these changes between raw materials to enrich the discussion.
Specific comments:
L23. all Latin names must be in italics
L26. The ratios is not well explicated. Is alfalfa:mulberry? Please explain it better.
L28. It has a relationship between in vitro and in vivo?
L26. In vitro in italics. Please see all names.
L28. Please see comments on L 23.
L70. Please add a space.
L137. The data was not checked for normality? kurtosis? Can anova be applied? If it has been done it must be specified, if it has not been done it should be done.
L155. Please explain DM and FM.
L155. TN is not refered in the Table 1.
L168. What is TMR? You should explain it.
L201. Table is in two pages.
L215. Point after tittle (among many).
L310. Is the only moment when you talk about amino acid. You should expand the information on this side a lot, since even the title itself talks about protein.
L349. Please, explain in more depth the in vivo correlation in vitro, as it is what really matters in production.
Author Response
Dear editor and reviewer,
We have answered the questions and corrected the mistakes in manuscripts who reviewer raised. Please download the attachment.
Kind regards,
Rongrong Li
