Protein Identification of Seminal Plasma in Bali Bull (Bos javanicus)
, Göran Andersson 2, Herry Sonjaya 3,*, Raden Iis Arifiantini 4, Syahruddin Said 5, Hasbi Hasbi 3, Tulus Maulana 5 and Abdullah Baharun 6
Round 1
Reviewer 1 Report (Previous Reviewer 2)
The manuscript titled Protein identification of seminal plasma in Bali Bull by Iskandar and co-workers has been revised twice. I have been clear in previous reviews that this is an observational study and any inference into function or fertility has not been established. To be fair to the reader all reference to “fertility biomarkers” must be removed—this study has not in any way established the role of these proteins in fertility. Certainly, they can be discussed and previously established roles of these proteins should be noted, but it is not an outcome of this study.
Specific suggestions
Line 19. Add “[inferred] function of these seminal plasma proteins…”
Line 19-20. Add “We [identified] six protein that [have been previously tied] with fertility”
Line 20 -21. This was not established in the current study. These proteins were identified in the current study and their functional role was in no way established. Revise to reflect the study.
Line 34. A functional reproductive role was not established in the current study. Based on what are these proteins correlated with fertility? Revise to reflect the results of the current study.
Line 37. Delete “and its potential as a biomarker of fertility”
Line 39 -40. Fertility or any measure of function was not established in this study. This sentence must be deleted.
Line 56. Comma between “expensive” and “because” is unnecessary –I’m pretty sure.
Line 62. Delete “yet”
Line 65-68. Sentence is long and confusing. Revise for clarity. I think sperm quality is more associated with fertility rather than the other way around. Suggest “Sperm quality is essential for bull fertility and seminal plasma proteins play an important role in sperm protection, capacitation, acrosome reaction, sperm-egg binding, fertilization, and early embryonic development.”
Line 68. Suggest “may” rather than “can also”
Line 70. Delete “on”
Line 76. Suggest “characterized” rather than “reported”
Line 80. Suggest “identified” rather than “found”
Line 113. Delete “Meanwhile”
Paragraph (lines 124 – 132). Reorganize for clarity. Delete sentence beginning… “lyophilization is the process…” I think the readers would be well aware of the process of lyophilization. The paragraph is disorganized and confusing since the process of freeze-drying is described as well as the resuspension. Suggest starting with the quantity of protein, the conditions of freeze-drying, followed by storage and resuspension.
Line 136. Suggest ‘for a final volume of 75 uL.”
Line 142. I think the impurities are in the sample and not the mass spec. Suggest “remove impurities from the sample”
Line 203 – 207 . Discussion and not results—move to discussion section.
Table 1. Suggest making it supplemental data, and not presented with the manuscript—I do not think it is referred to in the results.
Figure 2c. is lacking a title.
The discussion lacks reference to the published literature. There is no citations in the first paragraph. Much in the first paragraph requires citations also line 374, 350.
Line 371. Reference to [45] is duplicated.
Line 401. Suggest “Qu et al [55]…”
Line 401. Do you mean “seminal plasma” not “sperm plasma”
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report (Previous Reviewer 1)
The authors did a good job addressing all the raised issues and the manuscript is in my opinion now eligible for publication.
Author Response
Thank you very much
Round 2
Reviewer 1 Report (Previous Reviewer 2)
The revised manuscript is improved with the acknowledgement that this study is limited and there is no measure of fertility in this study.
Line 29 - 34. Long sentence--suggest revising. Perhaps "Proteins x, Y, Z, identified in seminal plasma of Bali bulls are identified as important for fertility in Bos Taurus. These proteins may prove to be important biomarkers of fertility in Bali bulls."
The sentence that follows is incomplete. Suggest "These proteins are important for reproductive function with function which include x, y, z."
Line 45. "Bali cattle [are] the most popular..."
Line 67. Delete "levels"
Author Response
Point 1: The revised manuscript is improved with the acknowledgement that this study is limited
and there is no measure of fertility in this study.
Response: Thank you very much. It was your valuable and insightful comments that led to possible
improvements in the current version.
Point 2: Line 29 - 34. Long sentence--suggest revising. Perhaps "Proteins x, Y, Z, identified in
seminal plasma of Bali bulls are identified as important for fertility in Bos Taurus. These proteins
may prove to be important biomarkers of fertility in Bali bulls."
Response: Thank you very much. We have made revisions accordingly [Page 1, Line 31-35].
Point 3: The sentence that follows is incomplete. Suggest "These proteins are important for
reproductive function with function which include x, y, z."
Response: Thank you very much. We have made revisions accordingly [Page 1, Line 41].
Point 4: Line 45. "Bali cattle [are] the most popular..."
Response: Thanks for your kind reminders. We revised the word [Page 2, Line 53].
Point 5: Line 67. Delete "levels"
Response: Thank you to the reviewer for pointing this out. We have removed
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
Please see the enclosed PDF file.
Comments for author File: Comments.pdf
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 2 Report
The manuscript titled Protein identification f seminal plasma in Bali Bull (Bos javanicus) by Iskander and co-workers collected semen from 10 bulls and using LC-MS/MS protocols to identify 94 seminal plasma proteins. Since no measure of fertility was determined for these bulls, it cannot be established that any of these proteins are associated with fertility. Certainly some of these proteins have been identified as important by other studies, but not in this study or for Bali bulls. The experimental design of this study is not hypothesis based and while it is observational. Proteins were identified in the seminal plasm and some of these have been identified to semen quality or sperm function. This is not unexpected.
Other considerations
Line 23, 46. Cows return to “estrus” not “lust”.
Line 32-33 and 386. LC-MS/MS is a method and cannot be used as a marker of male fertility.
Line 75. I think this would be characteristics of thawed semen.
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Reviewer 3 Report
Simple summary: in line 18, change LS-MS to LC-MS.
Abstract: no comments.
Introduction: line 69, add “semen plasma protein has been studied/ reported”, otherwise the sentence looks incomplete.
Line 74, “ bos … seminal plasma was analyzed”, add this or otherwise sentence looked incomplete.
Look for such incomplete sentences throughout the paper and correct.
Material and methods: line 125, 75 l means 75 Litre?
Results: line 213 sounds repeated.
Table 1, rows are not in harmony, please check
Discussion: no comments
Conclusion: no comments
Author Response
Please see the attachment
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors significantly modified the manuscript in accordance with my previous comments. I believe that the current version still needs a few minor corrections.
Suggested corrections are marked in the enclosed PDF file on the following lines:
· L34
· L47
· L61
· L66-67
· L121-122
· L242
· L278
Highlighted issues are briefly explained here:
· L74 and L76 are almost identical. Please omit one of them.
· L109: Please explain the reason for the use of such a high mass of protein.
· L126: Please briefly describe the protocol for lyophilization.
· L392: Please use uppercase letters (Ca2+).
The manuscript should be eligible for publication after corrections of above-mentioned issues.
Comments for author File: Comments.pdf
Reviewer 2 Report
The manuscript titled Protein identification f seminal plasma in Bali Bull (Bos javanicus) by Iskander and co-workers collected semen from 10 bulls and using LC-MS/MS protocols to identify 94 seminal plasma proteins. The revised manuscript is not improved. The study design is only observational and cannot establish that identified proteins in the seminal plasma of these bulls are associated with any measure of fertility. The results of this study do not support the stated conclusion that these 95 proteins are potential biomarkers of fertility. All that has been established is that Bos javanicus bulls express similar seminal plasma proteins that have been shown in other bulls. This is not unexpected.
