Characterization of Seminal Plasma Extracellular Vesicle MicroRNAs and Their Association with Boar Semen Quality During the Summer Season
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors identified a number of miRNAs associated with differences in boar semen quality during the summer season. Some comments are provided below:
Introduction: Please list the methods for assessing semen quality used in livestock breeding. Isolating extracellular vesicles and assessing their miRNA profile is a non-trivial task and requires sophisticated equipment. Therefore, it is doubtful that this approach will be adopted in livestock breeding.
Line 84: Please describe the animal groups studied. What were the inclusion criteria for each group? Describe the semen parameters in more detail.
Line 94: Please describe how extracellular vesicles were isolated. How was the number of EVs estimated? What volume of seminal fluid contains this number of EVs?
Figure 2b does not allow us to estimate the size distribution of the isolated EVs. Please provide NTA data.
The blot in Figure 2c is questionable. Unfortunately, the original images are not available.
Line 117. Please explain what the first two principal components (PC1 and PC2) are.
Figure 3c. Please list the 3rd and 9th miRNAs in the figure.
Line 186. Why were these specific miRNAs chosen? Please justify.
Author Response
Summary
We would like to thank the reviewers for their insightful and constructive comments. Your feedback has been instrumental in improving the quality and readability of our work. We greatly appreciate the time and effort you dedicated to reviewing our manuscript. We have carefully considered all your suggestions and incorporated your recommendations to improve the clarity of our study.
Point-by-Point Response to Comments and Suggestions for Authors
Reviewer 1
Comment 1: Introduction: Please list the methods for assessing semen quality used in livestock breeding. Isolating extracellular vesicles and assessing their miRNA profile is a non-trivial task and requires sophisticated equipment. Therefore, it is doubtful that this approach will be adopted in livestock breeding.
Response 1: We thank the reviewer for this insightful comment. In the revised Introduction, we have expanded the description of commonly used methods for assessing semen quality in livestock. These methods include computer-assisted sperm analysis (CASA) to assess sperm motility, morphology, and kinematic parameters, as well as flow cytometry–based analysis to evaluate sperm viability, acrosome integrity, mitochondrial membrane potential, and DNA fragmentation (lines 33-38). We agree with the reviewer that isolating extracellular vesicles and profiling their miRNA cargo are technically demanding and currently require specialized equipment. However, the primary aim of this study is not to propose immediate routine implementation in livestock breeding, but rather to identify novel molecular biomarkers that could be used to improve semen quality assessment and/or added to semen extenders. We have clarified this point in the revised manuscript (lines 85-86 and 87-94).
Comment 2: Line 84: Please describe the animal groups studied. What were the inclusion criteria for each group? Describe the semen parameters in more detail.
Response 2: We thank the reviewer for their insightful comments. Semen samples were collected from healthy, sexually mature Duroc breeding boars housed at a commercial boar stud and routinely used for artificial insemination. All animals were of comparable age and body weight, minimizing biological variability (lines 354-361). We did not use predefined experimental animal groups; however, semen ejaculates were categorized into two groups based on industry-standard semen quality assessments: “Passed” and “Failed”. Inclusion criteria for these groups were based on routine semen evaluation parameters (total motility and normal morphology) used in commercial artificial insemination programs. Ejaculates classified as “Passed” met the minimum commercial thresholds for artificial insemination, including high total motility (≥70%) and a high proportion of morphologically normal spermatozoa (≥70%). In contrast, “Failed” ejaculates did not meet these thresholds, exhibiting reduced total motility (<70%) and a low proportion of normal sperm morphology (<70%). These criteria ensured that the grouping reflected biologically and commercially relevant differences in semen quality. This has been addressed in lines 393-399.
Comment 3: Line 94: Please describe how extracellular vesicles were isolated. How was the number of EVs estimated? What volume of seminal fluid contains this number of EVs?
Response 3: We acknowledge the reviewer for their insightful comment. Extracellular vesicles (EVs) were isolated from 5 mL of boar seminal plasma by differential ultracentrifugation following standard protocols. Briefly, seminal plasma was subjected to sequential centrifugation steps to remove cells and debris, followed by ultracentrifugation to pellet EVs (lines 406-408).
The number of EVs was determined using nanoparticle tracking analysis (NTA) with the ZetaView® QUATT Particle Size Analyzer. For NTA measurements, EV samples were diluted 1:2000 in sterile distilled water to achieve an optimal particle concentration within the instrument’s detection range. A volume of 1 µL of the EV suspension was used for analysis. Based on the measured particle concentration obtained from NTA and accounting for the dilution factor, the total number of EVs was calculated and corresponds to the yield derived from the initial 5 mL of seminal plasma (lines 426-428). The concentration expressed as the number of SP-EV particles per milliliter (ml) is indicated in lines 111 and 114.
Comment 4: Figure 2b does not allow us to estimate the size distribution of the isolated EVs. Please provide NTA data.
Response 4: We have revised Figure 2 to provide the NTA size distribution in 2b.
Comment 5: The blot in Figure 2c is questionable. Unfortunately, the original images are not available.
Response 5: We have attached the original, uncropped blot images for assessment. Given the small size of EVs and limited sample availability, low levels of protein were loaded, which, together with our immunoblotting system, resulted in weaker signal bands that reflect the low protein concentration in boar SP-EVs. Nevertheless, the images in Figure 2c show most EV markers consistent with the EV characterization in this study.
Comment 6: Line 117: Please explain what the first two principal components (PC1 and PC2) are.
Response 6: This has been addressed in lines 138-142 and in the legend of Figure 3c.
Comment 7: Figure 3c. Please list the 3rd and 9th miRNAs in the figure.
Response 7: Figure 3c has been revised, and the unique miRNAs in Passed and Failed SP-EVs have been listed.
Comment 8: Line 186. Why were these specific miRNAs chosen? Please justify.
Response 8: The selected miRNAs were chosen for validation through RT-qPCR based on both their differential expression patterns and abundance identified in the miRNA sequencing dataset. Specifically, ssc-miR-7-3p was selected as a representative of upregulated miRNAs, while ssc-miR-205 and ssc-miR-802 were selected as representatives of downregulated miRNAs in Passed SP-EVs compared to Failed samples. Both the downregulated miRNAs were prioritized due to similar differential expression in boar SP-EVs isolated during the winter season in a similar study (https://www.mdpi.com/1422-0067/24/4/3194). In addition, ssc-let-7a was included because it was among the most abundant miRNAs detected in both groups (lines 211-212).
Comments on the Quality of Figures
Comment 1: Figures and tables must be improved.
Response 1: We have improved the quality of the figures where necessary.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAuthors have made a comprehensive comparison between passed and failed SP-EVs to identify some unique differentially regulated miRNAs that reflect their possible roles in physiological responses to changes in environmental conditions. It’s cool to understand that, like summer, there are some miRNAs that get dysregulated in winters as well, while winters are considered good in terms of fertility among boars. It would be of great importance to compare these identified miRNAs with the expressions during winter and summer season and identify the regulator to improve fertility during the summer.
The presentation of data needs some modification with better representation and clarity.
Fig 2a: Graph should be represented over Log at the Y axis. The current representation gives the impression that there is a very strong tendency to increase.
Fig 2c: The western blot image is hardly visible. If the Western blot images are so poor, it would be better to have the raw picture. There is a strange movement of the protein band for CD81 in the P-EVs lane and that does not correspond to the 26KDa (CD63). I am not very sure but when a protein in a lane moves almost all the proteins in that lane have similar movement, and since 23 KDa and 26KDa are very close to each other, I expect the band structure to be similar. Please reconfirm the blot represented for the result!
Authors should mention the n: no of samples analyzed in applicable figure legends.
Authors have listed miR-205 as a marker for seasonal variation. Among others, three downregulated DEMs, ssc-miR-205, 256 ssc-miR-802, and ssc-miR-9846-3p, were detected in both winter and summer boar SP-257 EVs, suggesting potential seasonal stability of these miRNAs. What does it mean by the statement seasonal stability? It would be important to know whether they increase, decrease, or remain consistent during the seasonal variation to better know the trend or importance.
However, authors have discussed the results comprehensively, but they have not reached any conclusion. Resulting in the addition of some data to the already available results on Boar samples. They have correlated their results with previously reported data and left everything for future research. It would be good if they could analyze some of them and compare them properly with the expression in winter. Like their data, there is much more data available; why not compare the data to find out the relevance of miRNA from their results, that changes during seasonal variation.
Author Response
Summary
We would like to thank the reviewers for their insightful and constructive comments. Your feedback has been instrumental in improving the quality and readability of our work. We greatly appreciate the time and effort you dedicated to reviewing our manuscript. We have carefully considered all your suggestions and incorporated your recommendations to improve the clarity of our study.
Point-by-Point Response to Comments and Suggestions for Authors
Reviewer 2
Comment 1: Authors have made a comprehensive comparison between passed and failed SP-EVs to identify some unique differentially regulated miRNAs that reflect their possible roles in physiological responses to changes in environmental conditions. It’s cool to understand that, like summer, there are some miRNAs that get dysregulated in winters as well, while winters are considered good in terms of fertility among boars. It would be of great importance to compare these identified miRNAs with the expressions during winter and summer season and identify the regulator to improve fertility during the summer.
Response 1: We thank the reviewer for this insightful comment. We have previously investigated boar SP-EV miRNA profiles during the winter season in a study published in 2023 (https://www.mdpi.com/1422-0067/24/4/3194). In that study, miRNA sequencing identified only three differentially expressed miRNAs: ssc-miR-205, ssc-miR-802, and ssc-miR-9846-3p, all of which were downregulated. Interestingly, two of these miRNAs (ssc-miR-205 and ssc-miR-802) were also identified in the present summer miRNA dataset, suggesting a potential role as seasonally responsive biomarkers. This overlap supports the hypothesis that specific SP-EV miRNAs may regulate, directly or indirectly, semen quality in response to environmental changes. This has been addressed in the Discussion in lines 283-286.
Comment 2: The presentation of data needs some modification with better representation and clarity.
Response 2: All figures have been revised to improve clarity and readability, especially Figures 1, 2, and 3.
Comment 3: Fig 2a: Graph should be represented over Log at the Y axis. The current representation gives the impression that there is a very strong tendency to increase.
Response 3: The Y axis for Figure 2a has been revised.
Comment 4: Fig 2c: The western blot image is hardly visible. If the Western blot images are so poor, it would be better to have the raw picture. There is a strange movement of the protein band for CD81 in the P-EVs lane and that does not correspond to the 26KDa (CD63). I am not very sure but when a protein in a lane moves almost all the proteins in that lane have similar movement, and since 23 KDa and 26 KDa are very close to each other, I expect the band structure to be similar. Please reconfirm the blot represented for the result!
Response 4: We thank the reviewer for their suggestion. We have revised Figure 2c by choosing other gel images to improve clarity and visibility. Below are the originals and the uncropped raw blot image. We agree that the close molecular weights of CD81 (~26 kDa) and CD63 (~23–26 kDa) may make the band patterns appear inconsistent. It is known that variability in band migration can arise from factors such as gel conditions, antibody specificity, and post-translational modifications, which can affect the molecular weight of observed bands.
Comment 5: Authors should mention the n: no of samples analyzed in applicable figure legends.
Response 5: The figure legends have been revised (lines 100-104, 107-108, 155, and 172).
Comment 6: Authors have listed miR-205 as a marker for seasonal variation. Among others, three downregulated DEMs, ssc-miR-205, ssc-miR-802, and ssc-miR-9846-3p, were detected in both winter and summer boar SP-EVs, suggesting potential seasonal stability of these miRNAs. What does it mean by the statement seasonal stability? It would be important to know whether they increase, decrease, or remain consistent during the seasonal variation to better know the trend or importance.
Response 6: By “seasonal stability,” we intended to indicate that the same miRNAs (ssc-miR-205, ssc-miR-802, and ssc-miR-9846-3p) exhibited a consistent pattern of downregulation in both winter and summer datasets when comparing Passed and Failed SP-EVs. Therefore, their stability refers to the reproducibility of their differential expression trend across seasonal conditions. We have revised the manuscript to clarify this point (lines 283-286).
Comment 7: However, authors have discussed the results comprehensively, but they have not reached any conclusion. Resulting in the addition of some data to the already available results on boar samples. They have correlated their results with previously reported data and left everything for future research. It would be good if they could analyze some of them and compare them properly with the expression in winter. Like their data, there is much more data available; why not compare the data to find out the relevance of miRNA from their results, that changes during seasonal variation.
Response 7: We thank the reviewer for their constructive comment. We agree that a direct comparison between seasonal datasets would provide valuable insights into the role of miRNAs in regulating semen quality under varying environmental conditions. This suggestion is our goal in the next study. Here, our primary objective was to characterize miRNA profiles in boar SP-EVs associated with semen quality during the summer season. The selected miRNAs for further RT-qPCR validation were chosen to confirm their differential expression patterns in both winter and summer, which will inform future studies, as suggested by the Reviewer. This concern is addressed at different levels in the manuscript (lines 211-217, 283-286, and 322-325).
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsI found the manuscript “Characterization of Seminal Plasma Extracellular Vesicle MicroRNAs and their Association with Boar Semen Quality During the Summer Season” interesting because the authors aimed to build an association between microRNA in boar seminal plasma extracellular vesicles and semen quality. This is relevant to the field of semen analysis and would help improve assessment accuracy. The originality level is average given that research has been conducted both in pigs and other species by other researchers.
The manuscript gives a good description of the methodology and uses appropriate references.
There is no dedicated section for conclusions but this not mandatory and the findings are well displayed in the Results section by employing several types of graphs which present the findings in an illustrative manner (I found the volcano plot particularly useful). This is followed by a thorough explanation for the functions of various microRNA in the Discussion.
However, the most important drawback of this manuscript is that it does not stress what the research brings new to this field, what specific gap in the field of extracellular vesicle microRNA analysis it addresses that has not been previously covered. In my opinion the authors need to correct this.
Author Response
Summary
We would like to thank the reviewers for their insightful and constructive comments. Your feedback has been instrumental in improving the quality and readability of our work. We greatly appreciate the time and effort you dedicated to reviewing our manuscript. We have carefully considered all your suggestions and incorporated your recommendations to improve the clarity of our study.
Point-by-Point Response to Comments and Suggestions for Authors
Reviewer 3
Comment 1: I found the manuscript “Characterization of Seminal Plasma Extracellular Vesicle MicroRNAs and their Association with Boar Semen Quality During the Summer Season” interesting because the authors aimed to build an association between microRNA in boar seminal plasma extracellular vesicles and semen quality. This is relevant to the field of semen analysis and would help improve assessment accuracy. The originality level is average given that research has been conducted both in pigs and other species by other researchers.
Response 1: We thank the reviewer for their positive assessment of our manuscript and for recognizing its relevance to the field of semen analysis.
Comment 2: The manuscript gives a good description of the methodology and uses appropriate references.
Response 2: We appreciate the reviewer’s feedback on the methodology and the use of appropriate references.
Comment 3: There is no dedicated section for conclusions but this not mandatory and the findings are well displayed in the Results section by employing several types of graphs which present the findings in an illustrative manner (I found the volcano plot particularly useful). This is followed by a thorough explanation for the functions of various microRNA in the Discussion.
Response 3: We acknowledge the reviewer’s comments. There is a Conclusion section at the end of the Materials and Methods section that summarizes the key findings and their implications (lines 523-528).
Comment 4: However, the most important drawback of this manuscript is that it does not stress what the research brings new to this field, what specific gap in the field of extracellular vesicle microRNA analysis it addresses that has not been previously covered. In my opinion the authors need to correct this.
Response 4: We have expanded the Introduction and Discussion sections to provide a more comprehensive interpretation of the novelty of our study and its findings, with the aim of improving semen quality assessment and seasonal infertility diagnosis. This has been addressed in lines 80-94 and 354-361.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe implemented revisions have substantially strengthened the manuscript. All issues I previously identified have been resolved, resulting in a marked improvement in the text's overall quality. I have no further feedback.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have incorporated several changes that have improved the representation of data in this manuscript.
The author highlights a set of miRNAs that remains consistently downregulated during summer as well as winter. This represents seasonal stability with no variability to pinpoint any supportive role in the change in fertility during the season. My question stands to highlight the contrasting miRNA. It’s good to correlate the expression difference separately with previously reported data sets, but this does not help this manuscript to highlight the key differences to predict important miRNAs due to which there is decreased fertility in summer. Do 28 downregulated miRNAs identified in this study have any contrasting change with respect to winter study? Since the author has already published the winter study, they must clearly compare both studies. Except for miR-493-5p, not listed in the reported DEM list, none of the other reported had contrasting expression with season. The authors have listed most of the informational queries to be addressed in the following study. I feel strange that there is almost no clear variation shown in this article provided they have done a follow-up study as mentioned in the intro section. This article just stands as an informative report with almost no contrasting comparison with their previous study. I hope a comparative future study, as mentioned by authors, will give some better insight into the role of these miRNAs.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe manuscript can be published in its current form.
