Review Reports - First Insight into the Variation of the Milk Serum Proteome within and between Individual Cows

Round 1

Reviewer 1 Report

The aim of this research entitled “Variation in the milk serum proteome within and between individual cows” was to determine the variation between and within cows for their milk serum proteome using two datasets of the sample's milk.

This manuscript presents an interesting work, and the writing of the article is globally good with just some errors. However, I have a number of suggestions and comments to be addressed by the authors.

GENERAL COMMENTS

Introduction

In my opinion, the authors could improve the introduction by adding the recent developments in proteomic techniques in the bovine milk proteome.

Materials and Methods

In M&M, my main hesitation is the low number of samples, especially for dataset 1 which has only 4 cows. Furthermore, the sampling needs to be explained better.

Results

To better understand the results, I suggest adding a table with the significantly different whey proteins with respect to the variables and a table indicating the identified whey protein distribution on the biological functions and iBAQ value of the most dominant proteins

Discussion

The discussions need to be broadened and the authors must explain the observed differences. They also need to be supplemented with more recent and current references.

References

All references date back to the years 2000-2015, given the current topic, the bibliography and the paper must be updated with more recent works.

SPECIFIC COMMENTS

L 62: How did the milking take place? How was the milk collected? using an automatic milking system?

L 69: Please better specify the feeding; What is meant by "a combination"?

L 64-80 In the farm, were the cows selected for sampling divided into the barn based on productivity? (High, medium production ect). What was the average milk yield?

L 81: With what criteria did you choose the 17 samples from the complete dataset?

L 84: Has the milk composition analysis been performed? This is important when it comes to the lactation curve.

L 129: Please review citation 15.

L 145-147 Please rephrase the sentence better.

L 193: Feeding was the same for all cows (L 68). Please clarify this point.

L 204: Please review the description of the figure in the caption better. In any case, I suggest removing "The" (The number ect) and adding the number of cows (4). Also, add the tool/technique with which you have obtained this figure.

L 223: For figure2, please see the previous comment. Please also review the punctuation in the description.

L 240: For figure 3, please review the punctuation in the description and please improve the resolution, the writing cannot be read.

L 257: Please describe and explain figure 4 better. Attention to punctuation.

L 259-263: Attention, this result could be due to the small number of cows sampled for dataset 1 (only 4 animals). The authors should emphasize this aspect.

L 298-300: In this context and in the light of what has been stated, the authors should make a comparison with other dairy breeds.

L 304-306: How do the authors explain this aspect?

L 316: Please replace "…has been shown before" with "…has been previously demonstrated".

L 399: Check the point.

L 413: Check the point.

Author Response

GENERAL COMMENTS

Introduction

In my opinion, the authors could improve the introduction by adding the recent developments in proteomic techniques in the bovine milk proteome.

AU: Thanks for your suggestion. We have added the description of recent development in proteomics techniques in the bovine milk proteome in the introduction section, as shown in Line 50-53:

Proteomics techniques as an advanced approach for analysis of the relatively low abundant proteins has been widely used in bovine milk studies, including milk serum proteome, milk fat globule membrane proteome as well as their changes over lactation, during mastitis, between species, and after processing^7-8.

Materials and Methods

In M&M, my main hesitation is the low number of samples, especially for dataset 1 which has only 4 cows. Furthermore, the sampling needs to be explained better.

AU: With sample set 1, we would like to confirm that the milk proteome doesn’t change significantly over lactation. We have collected 5 timepoints for each individual cow and measured the proteome of these 20 samples. Although the number of cows was low, the result that not a single protein differed over lactation, while many differed among cows, is still a good indication that the variation in the proteome is more between than within cows. Furthermore, we have restructured the sample collection section as shown in Line 76-87.

Results

AU: Thanks for your suggestion. The intensity values of all samples, including biological function and subcellular location, are already mentioned in table S3. This has now been mentioned explicitly in line 268-269.

Discussion

The discussions need to be broadened and the authors must explain the observed differences. They also need to be supplemented with more recent and current references.

AU: Thanks for the comment. We have tried to broaden the discussion on the differences and added more recent references to the discussion section.

References

All references date back to the years 2000-2015, given the current topic, the bibliography and the paper must be updated with more recent works.

AU: We have updated the references as shown in the revised manuscript.

SPECIFIC COMMENTS

L 62: How did the milking take place? How was the milk collected? using an automatic milking system?

AU: The milk was collected by using an automatic milking system, which has been added to the revised manuscript (line 78-79).

L 69: Please better specify the feeding; What is meant by "a combination"?

AU: Thanks for your comments. “A combination” meant grass silage, maize silage, wheat, and pressed pulp being combined (added; line 84-85).

L 64-80 In the farm, were the cows selected for sampling divided into the barn based on productivity? (High, medium production ect). What was the average milk yield?

AU: Thanks for your comments. Indeed, the production of these selected cows was similar, with an average value of 23.52±3.12 kg/day.

L 81: With what criteria did you choose the 17 samples from the complete dataset?

AU: The milk of in total 32 cows was collected, from which we chose 17 cows based on their health status (SCC less than 10⁵), parity, and feeding type (added; line 92-95).

L 84: Has the milk composition analysis been performed? This is important when it comes to the lactation curve.

AU: Thanks for your comment. We have measured the composition of the milk of the 17 cows used for the individual variation analysis. The average fat content was 4.18±0.95g/100g, protein content was 3.06±0.64g/100mg, lactose content was 3.93±0.49g/100mg. The details of the 17 cows have added as Table S1.

L 129: Please review citation 15.

AU: We have added the citation in Line 148.

L 145-147 Please rephrase the sentence better.

AU: We have modified the sentence, as shown in Line 167-169.

L 193: Feeding was the same for all cows (L 68). Please clarify this point.

AU: Thanks for your comments. This was a mistake. We have removed feeding from the sentence.

AU: We have changed the caption of figure 1 (now figure 2) to “Number of quantified milk serum proteins in Samples set 1 (4 cows (A-D), 5 timepoints (1-5, being day 0, day 14, month 1, month 2 and month 3, label free proteomics)” as shown in the revised manuscript.

L 223: For figure2, please see the previous comment. Please also review the punctuation in the description.

AU: We have changed the caption of figure 2 (now figure 3) to “Number of quantified proteins in Samples set 2 (17 cows, 1 timepoint, dimethyl labeled proteomics)” as shown in the revised manuscript.

L 240: For figure 3, please review the punctuation in the description and please improve the resolution, the writing cannot be read.

AU: Thanks for your comment. We have revised caption of figure 3 (now figure 4), as shown below.

Hierarchical cluster of log2 ratios of quantified proteins in 17 cows. The log2 ratio was the abundance of one protein in each cow against the abundance of this protein in the pooled sample; when log2 ratio was 0, the color was black, when log2 ratio was more than 0, the color was red and when log2 ratio was less than 0, the color was blue. The darker the color intensity, the larger the difference between individual cows and the pooled sample. Proteins that couldn’t be quantified are labelled grey; Cluster A are highly varied immune-related proteins, Cluster B are relatively conserved nutrient transport proteins.

L 257: Please describe and explain figure 4 better. Attention to punctuation.

AU: We have revised the caption of figure 4 (now figure 5), as shown below.

Relative abundance distribution of the 12 most highly variable proteins among the 17 individual cows

L 259-263: Attention, this result could be due to the small number of cows sampled for dataset 1 (only 4 animals). The authors should emphasize this aspect.

AU: We have emphasized the influence of the small number of samples, as shown in Line 286-288.

L 298-300: In this context and in the light of what has been stated, the authors should make a comparison with other dairy breeds.

AU: We have added that the humoral immune response to bovine herpesvirus-1 infection was found to vary, being associated with genotype, in Irish dairy cattle, in Line 325-326.

L 304-306: How do the authors explain this aspect?

AU: We actually explained the reason why immune-related proteins varied more among individuals in the paragraph above it, Line 314-320.

L 316: Please replace "…has been shown before" with "…has been previously demonstrated".

AU: We have changed to “has been previously demonstrated” as shown in Line 345.

L 399: Check the point.

AU: We have revised this in the manuscript.

L 413: Check the point.

AU: We have revised this in the manuscript.

Reviewer 2 Report

General comments:

Generally, the paper is very well written and well structured.
The topic addressed is of great interest to the readers, both from a scientific and a practical point of view.
The introduction is very clear.
The experimental approach is correct but I believe that some elements need to be slightly modified or better clarified.
I therefore, consider the paper worthy of attention and of great interest to the readers of the journal.

Specific comments.

L 65-67: it would be appropriate to indicate the distance between the five samples. Furthermore, the distance from calving (or DIM) for each experimental subject should also be indicated, as a source of variability.
L 81: as many as 15 samples have been eliminated but the reason was not adequately explained. Better explain why they were collected but not used.
L 86: the method is adequately illustrated in lines 89-188. I think citation number 13 can be deleted.
L 188-193: statistical analysis is not appropriate. Theoretically, the sources of variability should be jointly analyzed. In addition, their interaction should also be tested.
In your case, then, the days of lactation (DIM) are not considered in any way.
The reason your study is limited to one-way analysis is the limited number of samples. While this is understandable for trial 1 (but should be explained in detail to readers), the same cannot be said for trial 2.
L 193: I cannot find the part of the material and methods section that describes the different diets

Author Response

AU: Thanks for your comments.

Specific comments.

AU: We have added the DIM of the five samples. The lactation days of the seventeen cows ranged from 112 to 247 days, as shown in the revised manuscript, Line 94.

L 81: as many as 15 samples have been eliminated but the reason was not adequately explained. Better explain why they were collected but not used.

AU: The milk of in total 32 cows was collected, from which we chose 17 cows based on their health status (SCC less than 10⁵), parity (2-5), and DIM (line 92-94).

L 86: the method is adequately illustrated in lines 89-188. I think citation number 13 can be deleted.
AU: Thanks for your comments. We have removed that citation.

L 188-193: statistical analysis is not appropriate. Theoretically, the sources of variability should be jointly analyzed. In addition, their interaction should also be tested.
In your case, then, the days of lactation (DIM) are not considered in any way.
The reason your study is limited to one-way analysis is the limited number of samples. While this is understandable for trial 1 (but should be explained in detail to readers), the same cannot be said for trial 2.

AU: Thanks for your comments. Indeed, one-way ANOVA used for sample set 1 is because of the relatively low number of cows. Based on the statistical analysis of sample set 1, we obtained the conclusion that the bovine milk serum proteome didn’t change significantly during mid lactation. Therefore, we collected sample set 2 from middle lactation for the individual variation analysis. We would like to focus only on the individual variations for sample set 2. We don’t take DIM into account for this second set, as we assume it is not causing variation, based on the results of sample set 1.

L 193: I cannot find the part of the material and methods section that describes the different diets

AU: Thanks for your comments. It was a mistake and we have removed feeding in the revised manuscript.

Reviewer 3 Report

DAIRY - MDPI, Referee’s Evaluation Report

MANUSCRIPT IDENTIFICATION: dairy-1475563

VARIATION IN THE MILK SERUM PROTEOME WITHIN AND BETWEEN

INDIVIDUAL COWS, (Original article)

Comments to Authors/Editor:

The paper of Zhang & colleagues aimed to quantify any possible variation between and within cows regarding their milk serum proteome. This manuscript falls within the scope of Dairy. The manuscript is sufficiently informative for the replication of the study. In general, the organization of the experiment seems to be well designed, yet, the English quality, grammar, and sentence structure must be improved. Indeed, the Abstract was written in a careless fashion, not a single value, location, the season of the year, and p-value among other important variables were included. The authors must re-write all this section. Introduction, L36, … “Milk proteins comprise relatively high abundant milk proteins…????; non-sense sentence, rewrite. Moreover, L36-44, …, abundant, abundant and abundant again; are the authors comfortable with such quantity of pleonasms??? Rewrite. While the authors clearly defined the objectives of the study, a working hypothesis was not considered; this is a must. L54-59, must be moved to the Material & Methods section. Please include the national dairy cattle inventory, and the contribution of this species to the national livestock sector; are dairy cows important from a productive, economic, or social standpoint; what breed was used??? In the M&M section, the authors must indicate the ethical approval number or code for the study from the home institution; the authors must confirm that the study was in line with international guidelines for the management of animals in research protocols; this is a must. Besides, I strongly recommended including a figure with the actual experimental protocol across time; this is a must. The breed used was included in L75, but must be included both, in the abstract and the objectives. I am really concerned regarding the number of cows and the range of parities in set-1. Please include in this part, the total number of samples collected between nov-mar, and adjust table 1; this is a must. Besides, both in the Abstract and M&M sections, the authors must include where the experiment was carried out (NL, WL), including the seasons and the main environmental indicators (i.e. temperature, humidity, radiation, photoperiod, etc.) along with the experimental period. The authors must include such information especially because this research includes physiological-related variables. Please define if the experimental samples were collected at the beginning, the middle or the last stage of the lactation. It is important to include the temperature-humidity index (THI) observed along with the experimental period, to better relate the physiologic responses of females to their environmental context. As mentioned, it is recommended to include information regarding the location, the main prevailing environmental conditions of temperature, humidity, radiation, photoperiod along with the experimental period. As mentioned, including a figure with the actual experimental protocol across time (i.e. a timeline of actions) should help the readership to better understand both the results and conclusions. The authors must indicate how the females were managed during the experimental period. Do the animals were managed as groups in the same or different corrals?? What was the area (i.e. m²per animal) for each treatment group? Were the animals group-feed or individually-feed?? Please clarify. Reagents, standards, and methods used are relevant and in accordance with the objectives of the study. Also, all the sampling techniques, laboratory methods, as well as response variables considered in the experiment are detailed and accurate, while in agreement with the objectives of the study. L161,… “Data analysis or Data analyses”…??? The experimental design, and statistical models, were not described well enough for the reader to understand how the experiment was carried out. Also, the authors must define if the response variables depicted a normal distribution or if they required an adjustment or transformation in order to perform the statistical analyses.

Regarding the Results section, the novelty value of the results is reasonable. L207, didn´t or did not??? Besides, the authors must include some kind of quantitative information aside from the P-values of the observed results regarding the response variables. Also, if no differences occurred among experimental groups (i.e. sampling time) for a defined variable, the authors must include the average value for such response variables observed in the study. Further, the inclusion of the “p-values” along with the word “significantly” is a pleonasm; just use one, not both, it is a pleonasm; correct accordingly along with the whole manuscript as well as Tables; this is a must. How different were the environmental conditions, besides the experimental unit number, between data set 1 and data set 2???, (i.e. year, month, location, nutrition, general management, parities, etc).

Results were shown in 1 Table & 4 Figures in the main manuscript, but supplementary information was also available from the study; this is a plus for the readership. Yet, the titles of figures and tables must be rewritten; the titles of tables must stand by themselves; titles must be rewritten. Besides, the authors must include the number of replicates used along with the experimental subsets; the authors must mention if the collection of samples was repeated across time within subsets. Regarding the Discussion section, at the beginning of the Discussion, I do strongly suggest initiating this section including the working hypothesis of the study. Authors must define if, with the obtained results, such a hypothesis is rejected or non-rejected. For this reason, the authors must include the working hypothesis prior to the objectives in the Introduction section of the manuscript. The authors must link, in a logical fashion, their main findings along with the discussion section, to compare & discuss and, thereafter, be able to propose some genetic, genomic, or physiologic explanations for such specific outcomes, considering previous similar studies from the scientific literature. In general, the authors made a fair interpretation of the main findings, and confront them with respect to the available scientific literature. The main outcomes of the study were soundly presented. The list of references cited in the manuscript is proper. This is an interesting study. As I mentioned, my main concern is the low replicates number; therefore, it could be important to include in the title something like “First insights” or “Preliminary results”. All the commented issues and requests should be clearly addressed by the authors; at this point, and based on the above comments, my pronouncement is that this manuscript requires moderate adjustments and corrections.

Author Response

AU: We have rewritten the abstract section, as shown in Line 14-28.

Introduction, L36, … “Milk proteins comprise relatively high abundant milk proteins…????; non-sense sentence, rewrite. Moreover, L36-44, …, abundant, abundant and abundant again; are the authors comfortable with such quantity of pleonasms??? Rewrite.

AU: Thanks for your comments. We have rewritten the sentence as shown in revised manuscript Line 38-40:

“Milk proteins comprise caseins and whey proteins, which contain low abundant immune-related proteins, such as immunoglobulins, proteins of the complement system, monocyte differentiation antigen CD14, osteopontin, clusterin, and alpha-1-antitrypsin.”

While the authors clearly defined the objectives of the study, a working hypothesis was not considered; this is a must.

AU: We have added the hypothesis in the introduction section, as shown in Line 57-59. Our hypothesis was that the milk proteome doesn’t change significantly during mid lactation of HF cows, however, they do show significant variation among individual HF cows.

L54-59, must be moved to the Material & Methods section.

AU: We have moved this part to the Material & Method section, as shown in Line 67-73.

Please include the national dairy cattle inventory, and the contribution of this species to the national livestock sector; are dairy cows important from a productive, economic, or social standpoint; what breed was used???

AU: Thanks for your comments. We have added this information in Line 54.

“Holstein Frisian is the dominant cow breed in the Netherlands.”

In the M&M section, the authors must indicate the ethical approval number or code for the study from the home institution; the authors must confirm that the study was in line with international guidelines for the management of animals in research protocols; this is a must.

AU: We have added this information, as shown in Line 72-73.

Besides, I strongly recommended including a figure with the actual experimental protocol across time; this is a must.

AU: We have visualized the experimental protocol in Figure 1.

The breed used was included in L75, but must be included both, in the abstract and the objectives.

AU: We have added Holstein Frisian cow in both abstract and objectives as shown in the revised manuscript on Line 18 and 57-59.

I am really concerned regarding the number of cows and the range of parities in set-1. Please include in this part, the total number of samples collected between nov-mar, and adjust table 1; this is a must.

AU: Thanks for your comments. In total, 20 samples were collected in sample set 1 and this information has been added in the revised manuscript, as shown on Line 82-83. The table 1 was adjusted.

Besides, both in the Abstract and M&M sections, the authors must include where the experiment was carried out (NL, WL), including the seasons and the main environmental indicators (i.e. temperature, humidity, radiation, photoperiod, etc.) along with the experimental period. The authors must include such information especially because this research includes physiological-related variables. Please define if the experimental samples were collected at the beginning, the middle or the last stage of the lactation.

AU: We have added this information in the revised manuscript, as shown in Line 110-111. Sample set 1 was analyzed in May 2012 and sample set 2 was analyzed in May 2013, which were all conducted at Wageningen University.

It is important to include the temperature-humidity index (THI) observed along with the experimental period, to better relate the physiologic responses of females to their environmental context. As mentioned, it is recommended to include information regarding the location, the main prevailing environmental conditions of temperature, humidity, radiation, photoperiod along with the experimental period. As mentioned, including a figure with the actual experimental protocol across time (i.e. a timeline of actions) should help the readership to better understand both the results and conclusions.

AU: Thanks for your comments. We do agree with the reviewer that the environmental conditions of temperature, humidity, radiation, photoperiod may influence the milk composition. All the samples were collected in winter. The sample set 1 was analyzed in May 2012 and sample set 2 was analyzed in May 2013, which were all conducted at Wageningen University.

The authors must indicate how the females were managed during the experimental period. Do the animals were managed as groups in the same or different corrals?? What was the area (i.e. m2 per animal) for each treatment group? Were the animals group-feed or individually-feed?? Please clarify.

AU: All cows were housed indoors, and were managed (including fed) as a single group, as added now in line 81-82.

Reagents, standards, and methods used are relevant and in accordance with the objectives of the study. Also, all the sampling techniques, laboratory methods, as well as response variables considered in the experiment are detailed and accurate, while in agreement with the objectives of the study.

AU: Thanks for your comments. We have added more detailed descriptions throughout the materials & methods section of the revised manuscript.

L161,… “Data analysis or Data analyses”…??? The experimental design, and statistical models, were not described well enough for the reader to understand how the experiment was carried out. Also, the authors must define if the response variables depicted a normal distribution or if they required an adjustment or transformation in order to perform the statistical analyses.

AU: Thanks for your comments. We have changed it to “analyses” throughout the whole manuscript. We have improved the description of the experimental design in the revised manuscript to better explain how these experiments help to test our objective and hypothesis. Transformation and normalization were applied during data analyses, of which the detailed description was shown in Line 199 and 201-203.

AU: Thanks for your comments. We have added this information in Table S2. There were two columns with ANOVA analyses. One is for the time points, the other is for differences among individual cows. It is clear from this table that there were no significantly different proteins found between time points; however, several significantly different proteins were found between individuals. The p-values were also added to Table S1. Although the two sample sets were not collected on the same farm, they were all collected during winter. The potential influence of season and environmental temperature on the milk proteome would thus have been avoided.

AU: Thanks for your comments. We have revised the figure and table captions as shown in the revised manuscript. We used dimethyl labeling for 17 individual cows analysis, all being compared to a pooled sample, without replicates.

Regarding the Discussion section, at the beginning of the Discussion, I do strongly suggest initiating this section including the working hypothesis of the study. Authors must define if, with the obtained results, such a hypothesis is rejected or non-rejected. For this reason, the authors must include the working hypothesis prior to the objectives in the Introduction section of the manuscript. The authors must link, in a logical fashion, their main findings along with the discussion section, to compare & discuss and, thereafter, be able to propose some genetic, genomic, or physiologic explanations for such specific outcomes, considering previous similar studies from the scientific literature. In general, the authors made a fair interpretation of the main findings, and confront them with respect to the available scientific literature. The main outcomes of the study were soundly presented. The list of references cited in the manuscript is proper.

AU: AU: Thanks for your comments. We have added the working hypothesis of the study in the beginning of the discussion, as shown in Line 280-282.

This is an interesting study. As I mentioned, my main concern is the low replicates number; therefore, it could be important to include in the title something like “First insights” or “Preliminary results”. All the commented issues and requests should be clearly addressed by the authors; at this point, and based on the above comments, my pronouncement is that this manuscript requires moderate adjustments and corrections.

AU: Thanks for your comments. We have added First insight in the title of manuscript “First insight into the variation of the milk serum proteome within and between individual cows”. We also revised the last paragraph of discussion and conclusion section, as shown in Line 351-352, 362.

Round 2

Reviewer 1 Report

I thank the authors who answered all my doubts and added all the corrections I suggested in the paper.
For this reason, I accept this new form of the manuscript.