Effect of Season on the Characteristics of Warmblood Stallion Spermatozoa Stored in a Liquid State at 5 °C
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe aim of this work is to analyse Warmblood stallion seminal characteristics during breeding and non breeding season when semen is stored at +5°C. seminal characteristics of motility (measured by CASA), acrosome integrity, plasma membrane integrity, Mitocondrial membrane potential, and adenosine triphosphate (ATP) content. the work is well written, experiment well structured, hypotheses and results well explained, discussion is relevant. I have just a few comments but for me the work is good, and it should be considered for publication after just very few modifications that are here addressed.
- Simple summary is clear, please adjust this phrase in order to add the information that these are results of this work (line 22-23): "Nevertheless, prolonged storage showed no significant differences in the quality of spermatozoa between the compared seasons."
- 2.1. Animals and ejaculate collections: a description of the inclusion and exclusion criteria of enrolment for animals and/or ejaculates should be added.
- sperm motility was evaluated under a microscope: if this motility parameter was assessed subjectively, this should be declared and /or explained (maybe saying as referred to reference which is reported?)
- 2.3.4. Mitochondrial membrane potential (MMP): please add some more information about wavelength of the filters used in order to evaluate spermatozoa at the fluorescence microscope
- Discussion: it is well written and it is easy to follow, just some comments.
- Why you call the conservation "in liquid state" ? I can't really understand the reason of this definition.
- Line 283-286: "The ejaculates collected in spring/summer (March-July, breeding season) were characterized by larger volume" Was there any difference in the total sperm count? Is this real or is it just a consequence of the decrease of volume of the ejaculate due to a decrease in the dilution by the seminal plasma production during the non breeding season?
- line 302-307: What's the point of this small paragraph? I think that, if the authors wanted to give further explanations on the ejaculate dilution and / or analysis post collection and / or to discuss the choice of using a "liquid state" (btw as already stated, for me it's hard to understand the importance of this definition given by authors), this should be linked to the previous section.
- In the conclusion, a comment about the impact of this study in the general topic related to seasonality and semen conservation in stallions could be added in order to address suggestion ot further studies.
Author Response
Reviewer comments and responses:
The aim of this work is to analyse Warmblood stallion seminal characteristics during breeding and non breeding season when semen is stored at +5°C. seminal characteristics of motility (measured by CASA), acrosome integrity, plasma membrane integrity, Mitochondrial membrane potential, and adenosine triphosphate (ATP) content. the work is well written, experiment well structured, hypotheses and results well explained, discussion is relevant. I have just a few comments but for me the work is good, and it should be considered for publication after just very few modifications that are here addressed.
Point 1: Simple summary is clear, please adjust this phrase in order to add the information that these are results of this work (line 22-23): "Nevertheless, prolonged storage showed no significant differences in the quality of spermatozoa between the compared seasons."
Response 1: The sentence in the simple summary was modified and the phrase "in this study" was added (line 23).
Point 2: 2.1. Animals and ejaculate collections: a description of the inclusion and exclusion criteria of enrolment for animals and/or ejaculates should be added.
sperm motility was evaluated under a microscope: if this motility parameter was assessed subjectively, this should be declared and /or explained (maybe saying as referred to reference which is reported?)
Response 2: The information that the ejaculates in which total sperm motility assessed under a microscope (immediately after semen collection) was above 70% were used for liquid storage, in accordance with the recommendations (Vidament et al. 2005, Egyptien et a. 2023).
The missing information was included in the methodological description (line 118).
Point 3: 2.3.4. Mitochondrial membrane potential (MMP): please add some more information about wavelength of the filters used in order to evaluate spermatozoa at the fluorescence microscope
Response 3: The description of fluorescence analyses was expanded to include the information on the wavelengths of the filters (lines: 157-160; 168-169; clear version). In normal mitochondria, JC-1 accumulates in the mitochondrial matrix as a polymer that exhibits strong orange-red fluorescence (Ex=488 nm, Em=595 nm). In mitochondria with low MMP, JC-1 cannot accumulate in the mitochondrial matrix and exhibits green fluorescence (ex=488 nm, em=530 nm). The Olympus BX41 fluorescence microscope, which uses a light source with ultraviolet (330–385 nm), blue (460–490 nm), and green (510–550 nm) excitation wavelengths, was used for fluorescence analyses in the study
Discussion: it is well written and it is easy to follow, just some comments.
Point 4. Why you call the conservation "in liquid state" ? I can't really understand the reason of this definition.
Response 4: As suggested, this phrase was corrected.
Point 5: Line 283-286: "The ejaculates collected in spring/summer (March-July, breeding season) were characterized by larger volume" Was there any difference in the total sperm count? Is this real or is it just a consequence of the decrease of volume of the ejaculate due to a decrease in the dilution by the seminal plasma production during the non breeding season?
Response 5: No significant differences in the total sperm count were found between seasons, which could be due to the fact that ejaculate volume was higher in the breeding season, whereas sperm concentration was higher in the non-breeding season. As noted by the Reviewer, ejaculate volume was lower in the non-breeding season due to a decrease in dilution caused by seminal plasma production. This observation was also made in previous studies (Shawki et al. 2021).
Point 6: line 302-307: What's the point of this small paragraph? I think that, if the authors wanted to give further explanations on the ejaculate dilution and / or analysis post collection and / or to discuss the choice of using a "liquid state" (btw as already stated, for me it's hard to understand the importance of this definition given by authors), this should be linked to the previous section.
Response 6: This part of the text focuses on explaining why the authors diluted the ejaculate in an extender immediately after collection in the stud. This procedure allowed for the preservation of high-quality sperm intended for storage at 5°C. Our previous study (unpublished data) has shown that ejaculates that were transported to the laboratory and then diluted in an extender (after 2-4 h of transport) had significantly reduced motility compared to those diluted at the place of semen collection (in the stud). This information, in my opinion, may be particularly important for horse breeders. For this reason, it was included in this passage, before the discussion on the assessment of the quality of sperm stored in a liquid state.
Point 7: In the conclusion, a comment about the impact of this study in the general topic related to seasonality and semen conservation in stallions could be added in order to address suggestion of further studies.
Response 7: The Conclusion section was expanded to include the information that stallion spermatozoa collected in non-breeding and breeding seasons are characterized by similar suitability for liquid storage, and that the fertilizing capacity of stored spermatozoa (including capacitation) should be examined in the future to confirm the present findings.
Thank you for taking the time to review the manuscript, and for the positive feedback.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors present a study to examine the influence of season on the quality and cold storage resistance of ejaculates from warmblood stallions stored at 5°C. The manuscript addresses the effects of both breeding and non-breeding seasons on different functional sperm parameters over time.
The research objective is clearly formulated and methodologically well designed, with a factorial design that adequately accounts for the interaction between season and storage time. The topic is scientifically relevant and timely. The study provides novel insights into the relationship between initial semen quality and its stability under refrigerated conditions, adding value to current knowledge in the field of equine reproduction.
I have a few comments that I would like to contribute, as well as point out some typos that went unnoticed in the manuscript.
Introduction
Line 47-48: For practical reasons, chilled semen is more recommended for equine AI than cryopreserved semen because it increases the chances of fertilization success [1–3].
References 1 and 3 are appropriate references for this statement, but reference 2 does not mention fertility, nor does it recommend the use of chilled semen, it evaluates the quality of chilled Icelandic horse semen, so it does not seem to be one of the most appropriate references.
2. Janett, F.; Sacher, K.; Hässig, M.; Thun, R. Quality of raw and of cold-stored semen in Icelandic stallions. J. Equine Vet. Sci. 2012, 32, 590–595. doi: https://doi.org/10.1016/j.jevs.2012.02.004.
Lines 48-50: Stallion semen is usually stored in a liquid state at a temperature of 4-5°C for up to three or four 49 days [3–4].
Reference 3 is cited correctly in the previous paragraph, but it may not be adequate in this sense, since it does not study semen cooled to 4-5º, but fresh, uncooled semen, and compares it with the use of frozen-thawed semen.
Material and methods
Was the reproductive history of the stallions known?
Please clarify if the stallions were under a regular semen collection routine during the study period, or if the ejaculates were collected exclusively for the purpose of this study. Furthermore, it would be beneficial to specify whether the same stallions participated in both the breeding and non-breeding seasons, and how many ejaculates each individual contributed in each phase. This information is important for assessing the consistency and balance of the experimental design.
Lines 116-177 and lines 119-120 are the same: Cooled spermatozoa were analyzed after 2 h (time – 0 h), 116 24 h, 48 h, 72 h, and 96 h of storage at 5°C.
Results
Some typographical errors have been noted in yellow
Discussion
Lines 273-276: Previous studies on the effect of season on the quality of stallion semen stored in a liquid state are limited, and those that are available have been conducted in other geographical locations. Furthermore, these studies mainly primarily focused on the assessment of motility, morphology and DNA integrity.
This text is based on references cited in the article; it would be useful to add them to this text to make it easier for the reader to identify the information.
Line 287: Similar observations were made previously by Shawki et al. [25] who found that ejaculate…….
Shawki et al [25] corresponds to reference 28
Lines 292 and 299 Janet et al. [15 ].
Please correct: Janett et al.
References
Some typographical errors have been noted in yellow
Comments for author File: Comments.pdf
The quality of the English throughout the manuscript is generally good and the text is understandable. However, there are occasional grammatical issues, redundancies and minor errors in phrasing that would benefit from careful proofreading by a native or fluent speaker of English. A thorough linguistic revision is recommended to improve clarity, consistency and overall fluency.
For example:“Previously studies have mainly primarily focused on the assessment of motility and DNA integrity.”
Previously studies
Previous studies have primarily focused on......
Author Response
Response to Reviewer 2 Comments
The authors present a study to examine the influence of season on the quality and cold storage resistance of ejaculates from warmblood stallions stored at 5°C. The manuscript addresses the effects of both breeding and non-breeding seasons on different functional sperm parameters over time.
The research objective is clearly formulated and methodologically well designed, with a factorial design that adequately accounts for the interaction between season and storage time. The topic is scientifically relevant and timely. The study provides novel insights into the relationship between initial semen quality and its stability under refrigerated conditions, adding value to current knowledge in the field of equine reproduction.
I have a few comments that I would like to contribute, as well as point out some typos that went unnoticed in the manuscript.
Introduction
Point 1: Line 47-48: For practical reasons, chilled semen is more recommended for equine AI than cryopreserved semen because it increases the chances of fertilization success [1–3].
References 1 and 3 are appropriate references for this statement, but reference 2 does not mention fertility, nor does it recommend the use of chilled semen, it evaluates the quality of chilled Icelandic horse semen, so it does not seem to be one of the most appropriate references.
- Janett, F.; Sacher, K.; Hässig, M.; Thun, R. Quality of raw and of cold-stored semen in Icelandic stallions. J. Equine Vet. Sci. 2012, 32, 590–595. doi: https://doi.org/10.1016/j.jevs.2012.02.004.
Response 1: We agree with this comment. The reference to a study by Janett et al. was removed. Another reference has been added (lines: 418-419, clear version).
Point 2: Lines 48-50: Stallion semen is usually stored in a liquid state at a temperature of 4-5°C for up to three or four 49 days [3–4].
Reference 3 is cited correctly in the previous paragraph, but it may not be adequate in this sense, since it does not study semen cooled to 4-5º, but fresh, uncooled semen, and compares it with the use of frozen-thawed semen.
Response 2: Thank you for pointing this out. Reference 3 was removed (line 51).
Material and Methods
Point 3:
Was the reproductive history of the stallions known?
Please clarify if the stallions were under a regular semen collection routine during the study period, or if the ejaculates were collected exclusively for the purpose of this study. Furthermore, it would be beneficial to specify whether the same stallions participated in both the breeding and non-breeding seasons, and how many ejaculates each individual contributed in each phase. This information is important for assessing the consistency and balance of the experimental design.
Response 3: The history of the stallions from which semen was collected for testing was documented by an entry in the stud book or in the register of stud farms mentioned in the manuscript. The state-owned stud farm in Łąck is a well-known breeding center and a mating point that meets all the criteria set for such centers. The other company, Marek Romanowski’s Stallion Farm, is also involved in the sale of semen; therefore, the owner must follow certain rules and keep records of stallions used for breeding. Ejaculates were collected systematically (once or twice a week) from each stallion during the breeding season and outside the breeding season (line 89, clear version). Semen from selected stallions was intended for sale to domestic and foreign breeders. For the purpose of the study, the stud farms provided us with two ejaculates from a stallion (n=8) during the breeding season (April - July) and two- three ejaculates from a stallion (n=8) during outside the breeding season (September - December) (lines: 93-96, clear version). The initial assessment of semen quality included parameters such as sperm motility, ejaculate volume, and sperm concentration, and it was performed at the stud farms.
Point 4: Lines 116-177 and lines 119-120 are the same: Cooled spermatozoa were analyzed after 2 h (time – 0 h), 116 24 h, 48 h, 72 h, and 96 h of storage at 5°C.
Response 4: Thank you for this comment. The redundant information was removed.
Results
Point 5: Some typographical errors have been noted in yellow
Response 5: The letters in the tables (presenting the results of the study), denoting significant differences between the compared parameters, were corrected as indicated.
Discussion
Point 6:
Lines 273-276: Previous studies on the effect of season on the quality of stallion semen stored in a liquid state are limited, and those that are available have been conducted in other geographical locations. Furthermore, these studies mainly primarily focused on the assessment of motility, morphology and DNA integrity.
This text is based on references cited in the article; it would be useful to add them to this text to make it easier for the reader to identify the information.
Response 6: The relevant references cited in the article were added to support the above statements. (line 281, clear version).
Point 7: Line 287: Similar observations were made previously by Shawki et al. [25] who found that ejaculate…….
Shawki et al [25] corresponds to reference 28Line 91-95, please clarified it in details instead of citation.
Response 7: Thank you for this comment. The number of the reference was corrected (28 instead of 25).
Point 8: Lines 292 and 299 Janet et al. [15 ].
Please correct: Janett et al.
Response 8: The name of the cited author was corrected (line 304, clear version )
References
Point 9:
Some typographical errors have been noted in yellow
Response 9: The marked typographical errors were corrected.
peer-review-45264616.v1.pdf
Comments on the Quality of English Language
Point 10: The quality of the English throughout the manuscript is generally good and the text is understandable. However, there are occasional grammatical issues, redundancies and minor errors in phrasing that would benefit from careful proofreading by a native or fluent speaker of English. A thorough linguistic revision is recommended to improve clarity, consistency and overall fluency.
For example:“Previously studies have mainly primarily focused on the assessment of motility and DNA integrity.”
Previously studies
Previous studies have primarily focused on.
Response 10: As recommended by the Reviewer, the manuscript has been revised by a professional translator and a native English speaker to eliminate grammatical errors and stylistic inconsistencies, and to improve overall readability and clarity of presentation (please see the attached certificate).
Thank you for taking the time to review the manuscript and for all insightful comments and valuable remarks.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article “Effect of Season on the Characteristics of Warmblood Stallion Spermatozoa Stored in a Liquid State at 5°C” by Dziekońska et al., is intended to prove that the spermatozoa of warmblood stallions collected outside the breeding season are more resistant to low temperature than those collected during the breeding season; however, some issues should be pointed out:
1.- Authors should provide evidence of at least one parameter of capacitation: [Ca2+]i, pHi or evaluate the capacitation using CTC instead of just evaluating acrosome integrity, to prove that temperature is not affecting this important fertility parameter
2.- Line 172. Using JC-1 to evaluate MMP is correct; however, I recommend use a spectrofluorometer or cytometer to quantitatively evaluate this dye
3.- Line 208. Please, instead using tables to show your results of TMOT, PMOT, and all the kinematic parameters of sperm motility (VAP, VSL, VCL, etc), use graphs, you have enough information to show to the readers a tendency of the temperature effect.
4.- Line 370. Please add to your conclusion if the sperm from non-breeding season is able to perform hyperactivation or not comparing to previous kinematic parameters of sperm motility reports about your animal model
Author Response
Response to Reviewer 3 Comments
Point 1: The article “Effect of Season on the Characteristics of Warmblood Stallion Spermatozoa Stored in a Liquid State at 5°C” by Dziekońska et al., is intended to prove that the spermatozoa of warmblood stallions collected outside the breeding season are more resistant to low temperature than those collected during the breeding season; however, some issues should be pointed out:
1.- Authors should provide evidence of at least one parameter of capacitation: [Ca2+]i, pHi or evaluate the capacitation using CTC instead of just evaluating acrosome integrity, to prove that temperature is not affecting this important fertility parameter.
Response 1: Unfortunately, the capacitation of stallion spermatozoa stored in a liquid state was not assessed in this study. We agree with the Reviewer that additional parameters, including capacitation-related factors, could be analyzed, and this issue was addressed in the Discussion section (lines: 375-379, clear version).
Point 2: 2.- Line 172. Using JC-1 to evaluate MMP is correct; however, I recommend use a spectrofluorometer or cytometer to quantitatively evaluate this dye.
Response 2: In this study, the amount of dye was not assessed, but the percentage of live sperm with active mitochondria (spermatozoa with orange inserts and green heads) in the entire study population was determined. Both a fluorescence microscope and a flow cytometer can be used to separate different sperm populations. In this study, a fluorescence microscope equipped with a range of filters was used to assess mitochondrial efficiency and plasma membrane integrity. We agree with the Reviewer that the use of a flow cytometer in fluorescence analysis would also be a good solution as it would significantly shorten the time of analysis and support the analysis of a much larger sperm population. Nevertheless, both methods are recommended and their results allow for a reliable assessment of the analyzed structures. We also discussed the advantages and disadvantages of these two methods in our previous study (Dziekońska et al. 2023).
Point 3: 3.- Line 208. Please, instead using tables to show your results of TMOT, PMOT, and all the kinematic parameters of sperm motility (VAP, VSL, VCL, etc), use graphs, you have enough information to show to the readers a tendency of the temperature effect.
Response 3: In our opinion, the tables presented in the manuscript are clear, but as suggested by the Reviewer, Table 3 data, i.e. the kinematic parameters of sperm motility, were presented in graphical form (Figure 1).
Point 4: 4.- Line 370. Please add to your conclusion if the sperm from non-breeding season is able to perform hyperactivation or not comparing to previous kinematic parameters of sperm motility reports about your animal model. 1.
Response 4: Sperm collected outside the breeding season, compared to those collected during the breeding season, were characterized by higher values of the kinematic parameters of motility, which could indicate that a certain population of sperm may maintain a motility pattern similar to hyperactive. However, further research is needed to confirm this hypothesis. Sperm hyperactivation can be assessed by establishing the minimum thresholds for curvilinear velocity (VSL) and lateral head displacement and the maximum threshold for linearity. These thresholds vary depending on the CASA system and the animal species. Such analyses were not included when designing the experiment. To test whether sperm collected outside the breeding season are capable of hyperactivation, additional experiments should be conducted under conditions that induce capacitation and hyperactivation. Based on the analyses performed in the present study, it is not possible to determine unequivocally whether sperm collected outside the breeding season are capable of hyperactivation. Nevertheless, the above comment is very valuable as it indicates that the research should be continued and should include the assessment of capacitation and hyperactivation (lines: 388-389, clear version).
Thank you for taking the time to review the manuscript, and for all insightful comments.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsNo further comments
