Effect of eCG on Terminal Follicular Growth and Corpus Luteum Development and Blood Perfusion in Estrous-Synchronized White Lamphun Cattle

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript titled “Vascular Characteristics of the Corpus Luteum and Dominant Follicles in White Lamphun Cattle, an Indigenous Species, after PMSG Treatment: Implications for Luteal and Follicular Development”, the authors aim to explore the correlation between the vascularity of the corpus luteum (CL) and follicles with their development stage and timing after PMSG priming. The study seeks to enhance understanding of the response to PMSG stimulation on follicle and CL development through Doppler ultrasonography. However, the presentation and clarity of the content require significant improvement. Therefore, major revisions are necessary before considering the manuscript for publication in Animals.

Specific Points:

• The title is descriptive and effectively presents the study’s aim.
• The simple summary needs revision, as it currently resembles the abstract. It should be rewritten in a way that is accessible to a general audience.
• The abstract provides an overview of the study but lacks a conclusion based on the major findings.
• The introduction is well-written; however, the study's rationale needs to be strengthened. Simply focusing on Lamphun cattle's reproductive characteristics does not sufficiently justify the research.
• The Materials and Methods section is logically structured. However, there is a significant issue with the statistical model. The authors are examining two factors simultaneously—PMSG treatment and CL/follicle development over time—yet the comparison is made between the experimental and control groups. The statistical analysis should be revised to clarify how PMSG treatment influences CL and follicle development when monitored via ultrasound.
• The Results section should be revised following the updated statistical analysis. The follicular growth patterns in both the PMSG and control groups should be presented in a single graph, with follicle and CL growth on the Y-axis and observation time on the X-axis.
• The Discussion should be improved by addressing the study’s limitations and suggesting future research directions.
• The Conclusion is overly lengthy and should be revised to focus on the key findings.

Author Response

In reply to letter of journal manager dated February 21, 2025

Manuscript ID Animals-3476886

 

We have taken the comments from the reviewers and editor into account, and have made major revision as follows (responses are in the blue and italic font).

 

1) Comments of Reviewer #1:

In the manuscript titled “Vascular Characteristics of the Corpus Luteum and Dominant Follicles in White Lamphun Cattle, an Indigenous Species, after PMSG Treatment: Implications for Luteal and Follicular Development”, the authors aim to explore the correlation between the vascularity of the corpus luteum (CL) and follicles with their development stage and timing after PMSG priming. The study seeks to enhance understanding of the response to PMSG stimulation on follicle and CL development through Doppler ultrasonography. However, the presentation and clarity of the content require significant improvement. Therefore, major revisions are necessary before considering the manuscript for publication in Animals.

 

Specific points

(1) The title is descriptive and effectively presents the study’s aim.

        Thank you for the reviewer’s comment. We are pleased that you found the title descriptive and effective in presenting the study’s aim. However, in response to the reviewer’s suggestion, we have revised the manuscript to specify eCG instead of PMSG and have focused terminal follicular growth (TFG) and corpus luteum blood perfusion. These revisions can be found in the manuscript (revised version, Lines 1-4).

 

(2) The simple summary needs revision, as it currently resembles the abstract. It should be rewritten in a way that is accessible to a general audience.

Thank you for your comment. We appreciate your feedback regarding the simple summary. In response, we have revised the summary to ensure it is more accessible to a general audience (revised version, Lines 24-39).

       

(3) The abstract provides an overview of the study but lacks a conclusion based on the major findings.

        Thank you for your valuable comment. We have revised the abstract to include a conclusion that highlights the major findings of the study (revised  version, Lines 60-65).

 

(4) The introduction is well-written; however, the study's rationale needs to be strengthened. Simply focusing on Lamphun cattle's reproductive characteristics does not sufficiently justify the research.

        Thank you for your constructive feedback. We agree that the rationale for the study could be more clearly articulated. In the revised manuscript, we have strengthened the justification for our research by highlighting the unique reproductive challenges faced by Lamphun cattle and the potential benefits of improving fertility management in this indigenous breed. We also discuss how the findings could contribute to broader applications in cattle reproductive health and management practices (revised version, Lines 69-120).

 

(5) The Materials and Methods section is logically structured. However, there is a significant issue with the statistical model. The authors are examining two factors simultaneously—PMSG treatment and CL/follicle development over time—yet the comparison is made between the experimental and control groups. The statistical analysis should be revised to clarify how PMSG treatment influences CL and follicle development when monitored via ultrasound.

        Thank you for your comment, we agree with your feedback and have made revisions in the manuscript (revised version, Lines 237-251).

 

(6) The Results section should be revised following the updated statistical analysis. The follicular growth patterns in both the PMSG and control groups should be presented in a single graph, with follicle and CL growth on the Y-axis and observation time on the X-axis.

        Thank you for your comment. In response, we have separated the follicular and CL growth data to demonstrate the significant differences between them. Additionally, we need to present the significance after the cows received eCG. Specifically, we found significant differences in both follicular growth and CL development after the injection of eCG.

 

For example, publications that do not include follicular and CL growth in a single graph.

Mahdavi-Roshan, H., A. Niasari-Naslaji, M. vojgani, and D. Nikjou. 2020. Size and number of corpora lutea and serum progesterone concentrations when administering two doses of eCG in an estrous synchronization treatment regimen for dairy cattle. Anim. Reprod. Sci. 2020:106620.

Vieytes, R. A., C. V. Gil, G. D. A. Gastal, and D. Cavestany. 2023. Equine chorionic gonadotropin administered on day 5 of a 7-days fixed-time artificial insemination program improves ovulation synchrony and corpus luteum function in anestrous beef cows. Theriogenology 2023: 62-68.

 

(7) The Discussion should be improved by addressing the study’s limitations and suggesting future research directions.

        Thank you for your suggestion. We agree with your comment and have revised the discussion section in the manuscript (revised version, pages 381-482).

(8) The Conclusion is overly lengthy and should be revised to focus on the key findings. 

        Thank you for your suggestion. We agree with your comment and have revised the discussion section in the manuscript (revised version, pages 483-494).

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Authors aimed to assess the influence of a low dose of eCG (400 IU) administered  3 days before CIDR removal on follicular and luteal growth and hemodynamics in White Lamphun multiparous cows treated for synchronized ovulations by using a 7-day CIDR-PGF protocol combined with a double administration of GnRH separated by 9 days. The information obtained from this Bos indicus breed, although preliminary in its nature, is useful and interesting, but is presented in a confused manner, including a wrongly described experimental design, confused description of results, etc. As a result, all sections need more development. Moreover, there are conceptual pitfalls that need to be corrected as described in the separated file.

Comments for author File: Comments.pdf

Author Response

In reply to letter of journal manager dated February 21, 2025

Manuscript ID Animals-3476886

 

We have taken the comments from the reviewers and editor into account, and have made major revision as follows (responses are in the blue and italic font).

 

1) Comments of Reviewer #2:

Authors aimed to assess the influence of a low dose of eCG (400 IU) administered  3 days before CIDR removal on follicular and luteal growth and hemodynamics in White Lamphun multiparous cows treated for synchronized ovulations by using a 7-day CIDR-PGF protocol combined with a double administration of GnRH separated by 9 days. The information obtained from this Bos indicus breed, although preliminary in its nature, is useful and interesting, but is presented in a confused manner, including a wrongly described experimental design, confused description of results, etc. As a result, all sections need more development. Moreover, there are conceptual pitfalls that need to be corrected as described in the separated file.

Title comment:

(1) It must be revised because the present title (1) does not described well the aim of the study (terminal follicular growth is also an information important to gather; and Doppler image was only a component of the CL functional assessment and was not used in follicle assessment); and (2), as authors stated rightly, terminal follicular development and CL development is a functional continuum that consistently needs to keep in the same order (first follicle and then, as a result of the follicular development promoted by eCG, the CL development). eCG does not have an effect on CL development directly because is administered in a small dose, 3 days before the end of the CIDR treatment. Finally, replace PMSG (an old and inappropriate term) by eCG.

        We have revised the title in response to comment, focusing on terminal follicular growth and corpus luteum blood perfusion in White Lamphun cattle, as indigenous species, after eCG treatment: Implications for follicular and luteal development (revised version, Lines 1-4).

 

Simple summary:

(2) Lines 26-29: Instead of talking of hormonal regime (a general term), authors must described the experimental approach for assessing the eCG treatment. Authors used a protocol to synchronize ovulations but also to synchronize the terminal follicle development in both groups that allowed them to evaluate comparable follicle populations.

        Thank you for your comment. We agree with your suggestion and have revised the manuscript to clarify the experimental approach used for assessing the eCG treatment. The study used a protocol for ovulation synchronization and to synchronize terminal follicle development in both groups, ensuring that comparable follicle populations were evaluated (revised version, Lines 24-39).

 

(3) Line 30: Replace DF growth by follicular growth or terminal follicular growth as DF is a stage of this development. Most probable, due to the protocol, eCG affected the growth of the selected follicle that ended in a larger DF. Consider this point throughout the manuscript.

        Thank you for the reviewer’s comment. We agree with your suggestion and have revised the manuscript. We have replaced “DF growth” with “terminal follicular growth (TFG)” in abbreviation throughout the manuscript.

 

(4) Lines 30, and 31: Definitions are required for CL and CSA before using these terms.

        Thank for valuable comment from reviewer, we have added definitions for “corpus luteum (CL) and “cross-sectional area (CSA)” in the manuscript before using these terms (revised version, Lines 30-31).

 

(5) Lines 31-33: Doppler images only assesses irrigation not P4 secretion. Please refer to Elisa analysis for the last.

        Thanks to reviewer’s comment, we have revised the manuscript to clarify that Doppler imaging assesses vascularity and not P4 secretion. To accurately reflect P4 level, we have referred to the ELISA analysis for plasma P4 measurements (revised version, Lines 31-33).

 

(6) Lines 33-35: Replace Statistical analyses (also used before) by correlation analyses and include the association between Doppler and progesterone with CL area, as if significant, the last can replace both assessment, and it can be done in real time.

        Thank for your thoughtful comment, we have revised the manuscript to replace “statistical analyses” with “correlation analyses” as suggested and have included the association between Doppler imaging, P4 levels, and CL area. As you pointed out, if this association is significant, it may offer a more efficient, real-time assessment (revised version, Lines 33-39).

 

(7) Lines 35-36: Author statement is right but is better to put it in a more conditional manner as the study considered only 12 cows.

        Thank you for your helpful comment, we agree that the statement would be more appropriate in a conditional manner given the study’s small sample size. We have revised the sentence in the manuscript (revised version, Lines 38-39).

 

Abstract:

(8) Lines 38-39: Replace PMSG and define eCG in the Simple Summary section. Moreover, eCG is not used to induce ovulation in ruminants. Please delete the statement.

        Thank reviewer’s comment, we have replaced PMSG with eCG and have explained how eCG can be used, including delete the induction of ovulation in ruminants (revised version, Lines 40-41).

 

(9) Lines 39-43: The aims need to be described appropriately. The study was designed to assess the effect of adding eCG on terminal follicular and luteal development in indigenous cows treated for ovulation synchronization. Authors studied the follicular and luteal dynamics after eCG using structural (follicular and luteal diameters and luteal area; volume is not required) and functional markers (P4 profiles and luteal vascularization for CL development).   

        Thank you for your helpful comment, we have revised the manuscript to more appropriately describe the aims of the study following your suggestion (revised version, Lines 44-47).

 

(10) Lines 44-46: Follicle assessment was not considered in the description of the methodology. 

        Thank you for your comment. We have now included a description of the follicle assessment in the methodology section to ensure it is clearly outlined (revised version, Lines 49-53).

 

(11) Lines 46-48: Results on follicular development were not considered, and only the Doppler results were considered in the luteal assessment. Results in correlation analyses are described insufficiently.

        We have revised and added information about the connection between Doppler imaging, follicular dynamics, and the relationship between blood perfusion and P4 levels, offering a more detailed explanation of the findings (revised version, Lines 53-60).

 

(12) Lines 46-48: Please delete the statement “PMSG-treated group significantly enhanced CL vascularization which… Not only need better writing but also follicle development influences luteal development and not the way around (must be corrected). Add this observation to the description of luteal results.    

        We have deleted the statement following the comment and have added the observation to the description of luteal results (revised version, Lines 51-60).

 

(13) Line 47: Doppler assessment measures hemodynamics (blood perfusion, irrigation, etc.) not vascularization. Take it in consideration when results in correlation analyses are described later.

Thank you for your suggestion. We appreciate the clarification regarding Doppler assessment measuring blood perfusion rather than vascularization directly. We have revised the manuscript accordingly, using “blood perfusion” throughout.

 

(14) Line 52-53: The study did not assess follicular hemodynamics. Please correct this statement.      

We have removed the reference to follicular growth in the sentence and have focused on luteal development instead (revised version, Lines 62-63).

 

(15) Lines 52-58: Please limit conclusions to a 2-3-line paragraph with the main results linked to the aims of the study.  

Thank you for your comment. We have revised the manuscript as per your suggestion, limiting the conclusions to a 2–3-line paragraph (revised version, Lines 59-63).

 

Comments: This section must be rewritten.

Keywords: Please include eCG, replace follicular development by follicular dynamics, add corpus luteum development, replace vascularization by irrigation, remove indigenous breed, and I suggest adding Doppler analysis

        Thank you for your thoughtful suggestions. We have revised the manuscript in accordance with your comments (revised version, Lines 64-65).

 

Introduction

Comment:

The conceptual framework of this section has little relationship with the assessment of the effect of eCG on terminal follicular development and luteal development and functionality in White Lamphun cows. It is focused on the influence of CL on pregnancy and on follicular competence for fertility. Again, despite unsustainable statements (e.g., CL influence in oocyte competence for fertility (authors should be interested in the opposite, the influence of follicular development on CL development and function), and CL hemodynamics for pregnancy diagnosis), this section should be focused on the contribution of eCG on follicular development and by this way, CL development, and why authors think that this indigenous cows would respond in a particular manner to eCG treatments. Moreover, the aim of this study stated by authors at the end of this sections is different to that in the Simple Summary and Conclusions sections. The experimental design is intended to study the influence of eCG on follicular and luteal dynamics and no information was gathered in blood perfusion of ovulatory follicles. In this development, authors should consider the information gathered by GA Perry and colleagues who have been working in this topic for a decade. The present view should have been described, and suggests that not only there is a lack of influence on ovulatory follicle diameter and estradiol production on CL development (McLean et al., 2022; Perry et al., 2023), but also (as described by Wiltbank et al (2014; ref: 3) in a respected metanalysis on the topic), that P4 concentration has little correlation with fertility or pregnancy losses provided that there is sufficient blood concentrations, and that the only moment that there is a significant association with fertility (and weak) is during early CL development (P4 concentration in days 4-7). In a point for authors, follicle size has a significant influence on luteal development, functionality, and fertility when ovulations are synchronized by adding GnRH, as in this study (see GA Perry et al review in Animal Reproduction, 2023).

        Thank reviewer’s comment, we have revised the introduction section throughout the manuscript following your suggestion.

 

Conclusion in this section: Must be rewritten

        We have revised the conclusion in our manuscript (revised version, Lines 483-494).

 

Materials and Methods

Comment:

This section was reasonably written despite some details that will be described. A main pitfall is the wrongly description of GnRH (1) administration in the protocol for hormone administration, both in the text but also in Fig 1 description. Authors should follow the protocol depicted in Fig.1(very clear figure), the only that is right in GnRH (1), but it is wrong in GnRH (2) (should be day 9).

        Thank you for reviewer’s comment. We have addressed three points in the materials and methods section of the manuscript (revised version, Figure 1) as follows.

        1) The protocol for the second dose of GnRH injection was changed from day 10 to day 9.

        2) The term “DF diameter” has been changed to “Terminal follicular diameter (TFD)”.

        3) The term “PMSG-treated group” has been changed to “eCG-treated group”.

 

(16) Line 109: Replace provided continuously both day and night by provided ad libitum as described below in the text.  

        We have revised the manuscript by replacing “provided continuously both day and night” with “provided ad libitum” (revised version, Line 133).

 

(17) Line 115: Delete White Lamphun because it was defined before. The same for Lines 117 118.

        We have deleted 'White Lamphun' following the reviewer’s comment (revised version, Lines 139-142).

 

(18) Line 139: Replace Ovarian Structure Assessment and DF Diameter Measurement by Ultrasound assessment of follicular development and ovulation

        We have revised the manuscript by replacing “2.3 ovarian structure assessment and DF diameter measurement” with “Ultrasound assessment of follicular development and ovulation” (revised version, Line 163).

 

(19) Line 142: Delete greyscale

        We have revised the manuscript by deleting the term 'greyscale' (revised version, Line 166).

 

(20) Line 146: Not clear what do longest and shortest axes mean. Better to use the commonly used nomenclature (e.g., Ginther, 2016).

        Thank you for your comment, we agree that using the commonly accepted nomenclature for measuring the CL or follicle would improve clarity. Therefore, we have revised the manuscript to specify that the measurements were taken using the “longest diameter” and “shortest diameter” as per the standard terminology outlined by Gunther (2016) (revised version, Lines 170 and 184-185).

 

(21) Line 155: Replace Measurement of CL Diameter, CSA, and Volume by Ultrasound assessment of CL diameter and CSA (the volume trait brings little information and is not commonly used to characterize luteal development.

        Thank you for your comment. We have revised the manuscript following your suggestion (revised version, Line 179).

 

(22) Line 192: Delete to Evaluate CL Functionality (it is not required)

        We have deleted “to evaluate CL functionality” following your comment (revised version, Line 216).

 

(23) Lines 195-197: The handling of blood samples and the interval to plasma separation need to be considered.

        We have revised the sentence in the manuscript to clarify that, after collection, the blood samples were promptly centrifuged at 4°C at 800 × g for 15 minutes to separate the plasma, which was stored at –20°C until further analysis, in order to present the handling of blood samples (revised version, Lines 221-223).

 

(24) Line 201: There is a lack of consistency between the detection limit of the ELISA assay and the definition of functional CL. Please review the point.

        Following your comment, we have clarified this point in the revised manuscript to address any potential inconsistency between the assay's detection limit and the definition of functional CL (revised version, Lines 225-231).

 

Results

Comment:

Results are presented in a very confused manner, despite the beautiful images that need an increase in size. To increase precision on follicular dynamics and ovulation and luteal dynamics and hemodynamics, authors are encouraged to describe it in Tables (means and deviations) and include these images to have representative information. It is easier to interpret (e.g., LM Dysart et al, Doi.org/10.1016/ j.anireprosci.2021.106817). The same is demanded for describing the results from correlation analyses. Please consider the correlation between CSA, progesterone, and blood perfusion in CL. If they are strong, CSA can be used to get real-time information on CL function.

         Thank you for your constructive feedback. We appreciate your suggestion to improve the presentation of the results for better clarity. We have explained and added the values in the description of the results in each section related to the figures. We hope these revisions address your concerns and make the manuscript more readable and interpretable. Furthermore, in the correlation figure, due to the significant relationships among parameters, we have presented the correlation coefficient (r value) on the right Y-axis and indicated significance with an asterisk in the revised manuscript.

 

Discussion

Comment:

It needs to be rewritten, focusing on the main findings of this study, the assessment of how the study results compare with those in Bos indicus and Bos taurus both in the association between follicular and luteal development, and in CL hemodynamics. Important information should be considered, particularly the studies from GA Perry, M Smith, Pohler and colleagues, and in CL development and fertility, the information from reference 3 (Wiltbank et al., 2014 ) should also be considered.

        Thank you for your insightful feedback. We understand the need to focus the discussion more clearly on the main findings of the study and how these results compare with those from other breeds, such as Bos indicus and Bos taurus, particularly in relation to the association between follicular and luteal development, as well as blood perfusion of CL. In response to your suggestion, we have rewritten the discussion section to highlight the key findings from our study. We have emphasized how these results contribute to the understanding of follicular and luteal development in White Lamphun cattle, and how they compare with findings in Bos indicus and Bos taurus, particularly in terms of the associations between follicular growth, ovulation, and luteal function (revised version, Lines 381-482).

 

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

General comments:

This study evaluated the vascularization of the CL through doppler and relationships between DF diameter, CL size, and plasma P4 concentrations in multiparous White Lamphun cows. The study design is adequate for the objectives. The ovulatory induction protocol with eCG gives novelty to the study other than its application to the local breed.  There are two groups (n = 4 and n = 5, respectively); probably it is better to make this distinction abstract.  Overall, the study is well done in all the sections. Five very informative figures support M&M and Results sections. The discussion is adequate to the findings and supported by relevant literature. Conclusions are supported by the results.  No major issues were found, but some minor corrections and suggestions are made mainly to improve the readability of the paper.

 

Specific comments:

L42: I suggest adding the dose of PMSG in abstract.

L76, 86: Please insert a paragraph. Please check the manuscript for this issue to improve the readability of this manuscript.

L94: Please define abbreviations at first use. Check the manuscript.

L101-108: Please add more information about reproductive phase; parity, last calving and if there are in phase of suckling (i.e., cycling vs. anestrus).

L257-258: This sentence is part of discussion.

L263, 300: Please improve the quality (size) of these figures.

L282: Please add the unit to the values.

L286: It is ratio a percentage?

L326-327: This sentence is part of discussion.

L332: I suggest defining the right y-axis.

L359-369: I suggest empathizing with the relationships of CL between groups is probably due to the DF and CL diameters. Are CL characteristics mediated through DF?

Author Response

In reply to letter of journal manager dated February 21, 2025

Manuscript ID Animals-3476886

 

We have taken the comments from the reviewers and editor into account, and have made major revision as follows (responses are in the blue and italic font).

 

1) Comments of Reviewer #3:

This study evaluated the vascularization of the CL through doppler and relationships between DF diameter, CL size, and plasma P4 concentrations in multiparous White Lamphun cows. The study design is adequate for the objectives. The ovulatory induction protocol with eCG gives novelty to the study other than its application to the local breed.  There are two groups (n = 4 and n = 5, respectively); probably it is better to make this distinction abstract.  Overall, the study is well done in all the sections. Five very informative figures support M&M and Results sections. The discussion is adequate to the findings and supported by relevant literature. Conclusions are supported by the results.  No major issues were found, but some minor corrections and suggestions are made mainly to improve the readability of the paper.

 

Specific points

(1) L42: I suggest adding the dose of PMSG in abstract.     

        Thank you for the reviewer’s suggestion. We have addressed the dose of eCG in the abstract (revised version, Line 43).

 

(2) L76, 86: Please insert a paragraph. Please check the manuscript for this issue to improve the readability of this manuscript.

        Thank you for the reviewer’s suggestion. We have inserted the paragraph in the introduction and have made revisions in the introduction section to improve the readability of the revised manuscript (revised version, Line 68-120).

 

(3) L94: Please define abbreviations at first use. Check the manuscript.     

        Thank you for the reviewer’s comment. We have revised the line you mentioned in the introduction section and changed “DF” to “TF.” Therefore, we have defined the abbreviation (TF) at its first use and made it consistent throughout the manuscript.

 

(4) L101-108: Please add more information about reproductive phase; parity, last calving and if there are in phase of suckling (i.e., cycling vs. anestrus).

        Thank you for your suggestion, we have added the information about animals in the manuscript as much as possible (revised version, Lines 126-128).

 

(5) L257-258: This sentence is part of discussion.

        Thank you for your comment. Actually, we need to emphasize that we found this in each part of the results. Therefore, we still need to keep this part and excluded it from the discussion to prevent confusion (revised version, Lines 290-295).

       

(6) L263, 300: Please improve the quality (size) of these figures.

        Thank you for your suggestion, we have improved the quality of figures in the revised manuscript (revised version, Lines 296-298).

 

(7) L282: Please add the unit to the values.

        Thank you for your comment. We already have it in the manuscript (Lines 320-321).       

 

(8) L286: It is ratio a percentage?

        Thank you for your feedback. We have revised to prevent confusion. I have changed “ratio” to “proportion” (revised version, Lines 319 and 332), including the figure and explanation (revised version, Lines 334-345).

 

(9) L326-327: This sentence is part of discussion.

Thank you for your comment. Actually, we need to emphasize that we found this in each part of the results. Therefore, we still need to keep this part and exclude it from the discussion to prevent confusion (revised version, Lines 361-365).

       

(10) L332: I suggest defining the right y-axis.

Following your suggestion, we have defined the right Y-axis as the correlation coefficient (r) in Figure 5 (revised version, Line 366).

       

(11) L359-369: I suggest empathizing with the relationships of CL between groups is probably due to the DF and CL diameters. Are CL characteristics mediated through DF?

        We have revised and emphasized the relationship between terminal follicle (TF) and corpus luteum (CL) as much as possible to highlight the significant results of this study in the revised manuscript (revised version, Lines 381-482).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have thoroughly revised the manuscript and have adequately addressed all the raised queries. In its current form, the manuscript meets the required standards and is suitable for publication.

Author Response

Thank you for your valuable feedback and for recommending our manuscript for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The present manuscript has been improved; but it still requires maturation in various topic that are detailed in the attached file. As commented before, the information obtained from this Bos indicus breed, although preliminary in its nature, is useful and interesting, and as it weakest part is the small number of cows, author should counterbalance this shortcoming by providing a detailed analysis of the results, clear and precise outcomes, and sound conclusions.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Writing and grammar can be improved.

Author Response

In reply to letter of journal manager dated March 8, 2025-revised 2

Manuscript ID Animals-3476886

 

We have taken the comments from the reviewers and editor into account, and have made major revision as follows (responses are in the blue and italic font).

 

1) Comments and suggestions #2:

The present manuscript has been improved; but it still requires maturation in various topic that are detailed in the attached file. As commented before, the information obtained from this Bos indicus breed, although preliminary in its nature, is useful and interesting, and as it weakest part is the small number of cows, author should counterbalance this shortcoming by providing a detailed analysis of the results, clear and precise outcomes, and sound conclusions.

        We would like to thank the reviewer for their constructive feedback and for acknowledging the value of the preliminary data from the Bos indicus breed. We appreciate the suggestion to further mature various aspects of the manuscript, and we have carefully reviewed the detailed comments provided in the attached file.

 

2. Title comment #2:

Much better. I suggest replacing CL blood perfusion by CL development and blood perfusion, because the last is just an expression of a sound CL development that needs to be assessed by more functional markers after adding eCG. In addition, I suggest removing “as indigenous species” as it does not add neither value nor information to the conceptual framework built to manage TFD in this breed. Finally, I suggest removing also “implications for follicular and luteal development, as it is not clear what authors pretend. Therefore, I suggest “Effect of eCG on Terminal Follicular Growth and Corpus Luteum Development and Blood Perfusion in Estrous Synchronized White Lamphun Cattle”

        Thank you for your suggestion. We have agreed to revise the title of the manuscript, as reflected in the revised version (revised version, Lines 2-4).

 

3. Simple summary #2:

(1) Line 24: Precision is required. Replace “ovarian function” by terminal follicular growth (TFG) and corpus luteum (CL) development in…

        Thank you for the reviewer’s comment. We have revised the manuscript following your suggestion (revised version, Lines 24-25).

 

(2) Line 25: Remove “A total”.

        We have removed “A total” from the manuscript following your suggestion (revised version, Line 26).

 

(3) Line 26: Replace “vehicle-treated” by untreated-control a more explicit term.

        We have changed “vehicle-treated group” to “untreated-control group” in all sections where 'vehicle-treated' appears, following your suggestion in the revised manuscript.

 

(4) Lines 28-29: Replace “follicular growth” by TFG) and “terminal follicle growth” by TFG.

        We have replaced “follicular growth and terminal follicle growth” with “TFG” following your suggestion (revised version, Line 29-30).

 

(5) Lines 26-28: Replace “Both groups followed the same ovulation synchronization protocol, which included CIDR insertion, GnRH, and PGF injections. The eCG-treated group received an additional dose of eCG to stimulate follicular growth” by Both groups followed the same ovulation synchronization protocol, which included a 7-day CIDR, PGF and GnRH protocol, but the eCG-treated group received an eCG dose at day 4 to stimulate TFG.

        We have revised the manuscript following your suggestion (revised version, Lines 27-29).

 

(6) Line 29: Replace “The results” by Results; also “increased” by accelerated, as you measured follicular diameter after a period.

        We have revised the manuscript following your suggestion (revised version, Line 29).

 

(7) Lines 30-31: CLs are assessed clinically in terms of diameter, and area, but not in terms of volume; remove the last please.

        We have removed “CL volume” and replaced “CSA” with “luteal area” in all sections where “CL volume” and “CSA” previously appeared in the revised manuscript.

 

(8) Lines 34-35: The statement “suggesting that faster TFG leads to more robust luteal growth, higher P4 production” needs to be moved after the description of the result in lines 35-36 and fused with the concluding paragraph.

        We have moved the phrase “suggesting that faster TFG leads to more robust luteal growth and higher P4 production” and incorporated it into the concluding paragraph, following your suggestion (revised version, Lines 35-39).

 

4. Abstract #2:

(9) Lines 40-42: Please replace the statement “Equine chorionic gonadotropin (eCG) has been shown to improve reproductive performance in cattle, particularly in animals with compromised ovarian activity. eCG administration stimulates follicular growth and enhances CL function” by Equine chorionic gonadotropin (eCG) has been shown to improve reproductive performance in cattle by stimulating terminal follicular growth. There is no evidence that eCG has a direct influence on CL function when used in estrous synchronization protocols.

        We have made revisions following your suggestion (revised version, Lines 40-41).

 

(10) Line 43: Replace “on terminal follicular growth (TFG) and luteal development” by on TFG and its consequences in luteal development as discussed above.

        We have made the replacement following your suggestion (revised version, Line 42).

 

(11) Lines 49-51: The following statement is confused and needs review “The development and blood perfusion of the CL were monitored at various stages post-ovulation, with representative ultrasound images of follicles and the CL analyzed to assess changes in follicular growth, CL area, and CL blood perfusion”. The methodology needs to be consistent with the aims. TFG assessment needs precision and has the same importance than CL development in assessing the effects of eCG administration.

        We have revised and reviewed the methodology, focusing on TFG assessment and the effects of eCG administration (revised version, Lines 47-55).

 

(12) Line 53: Please describe the main results in follicular development before describing luteal development and perfusion. It is important to keep in mind that is the result in follicular function that modulate luteal development.

        We have described the results of follicular development before discussing luteal development and blood perfusion, following your suggestion (revised version, Lines 55-62).

 

(13) Lines 55-57: The following statement can be deleted because is repeated immediately after “Increased blood perfusion in the CL was closely correlated with elevated plasma P4 levels, emphasizing the role of follicular growth in driving luteal function”.

        We have deleted the statement as per your suggestion in the revised manuscript.

 

(14) Line 59: Please delete “Therefore”.

        We have deleted it following your suggestion in the revised manuscript.

 

(15) Lines 60-62: Replace “follicular growth was identified as a key factor influencing CL development. The eCG-treated group showed a notable improvement in blood perfusion, which is essential for optimal luteal development” by results highlighted the key role that eCG administration has on TFG and CL development and function in White Lamphun cattle.

        We have made the replacement following your suggestion (revised version, Lines 61-63).

 

(16) Lines 62-64: The paragraph can be deleted as it does not add value and is too generic. Authors need to remind that they only worked with 12 cows.

        We have deleted it following your suggestion in the revised manuscript.

 

5. Introduction #2:

(17) Lines 71-78: This paragraph can be deleted and replaced by the role that AI has in the production performance of White Lamphun herds. Avoid long paragraphs please.

        We have deleted and replaced content, focusing on the role of AI in production performance (revised version, Lines 70-87).

 

(18) Lines 79-80: Replace “Fixed-time artificial insemination (FTAI) has emerged as a valuable tool to address these issues” by Fixed-time artificial insemination (FTAI) procedures facilitate the use of AI as…

        We have made the replacement following your suggestion (revised version, Lines 85-87).

 

(19) Line 95: Replace “offer” by often. In addition, add LH pulse frequency to sustain TFG and ovulation.

        We have replaced the word, following your suggestion (revised version, Lines 100-101).

 

(20) Lines 99-109: The importance of TFG needs to be consider in this section as it determines the follicular and oocyte competence for fertility.

        We have added information highlighting the importance of TFG and its connection to follicular development and oocyte competence (revised version, Lines 108-112).

 

(21) Lines 106-109: Please review this paragraph replacing the present comparative view, by a view where Doppler add valuable functional information, as it measures blood perfusion. In a clinical setting we only use luteal area and progesterone secretion when dealing with groups of cows. Doppler does not add determinant information, not only because the critical point in fertility is the TFG performance, but also, as stated by Wiltbank et al (2014) in their meta-analysis of P4, P4 need to get enough concentrations in plasma early in the luteal phase to exert its beneficial effect on gestation.

          Thank you for your suggestion. We have removed the sentence in the revised manuscript.

 

(22) Lines 110-116: This study aims to study the effect of adding eCG in the estrous synchronization protocol on TFG and CL function, and the CL development is assessed by growth rate, blood perfusion and progesterone secretion. It is not a Doppler study. Therefore, the hypothesis must be related to the direct influence of eCG on TFG and on its consequences on CL function. Again, the TFG performance is the determinant factor in fertility and a sound TFG would determine the CL functional potential. Using Doppler US add value to the assessment.

        Thank you for your comment. We have revised the manuscript to focus on the effect of adding eCG and have edited the hypothesis to reflect the influence of eCG on TFG and its consequences on CL function (revised version, Lines 117-123).      

 

Comment: The context of this study needs improvement; it is a study directed to assess the effect of adding eCG to an estrous synchronization protocol for improving fertility after FTAI in this indigenous breed. The conceptual framework of this section still needs to integrate the TFG as a critical factor for fertility, more or at least as important than CL development and function. Hypothesis and aims need precision. Conclusion: Better but the section still needs maturation.

        Thank you for your suggestions and comments. We have carefully considered your valuable feedback and revised the manuscript to improve clarity. Please refer to the section of the Introduction in the revised manuscript.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     

6. Materials and Methods #2:

(23) Line 127: Please delete “an indigenous breed”

We have deleted it following your suggestion (revised version, Lines 129-130).

 

(24) Line 142: Replace “twelve multiparous cows (n=12)” by Experimental cows were… Please avoid repeating statements already described, including White Lamphun and indigenous cows.

        We have replaced “twelve multiparous cows (n=12)” with “experimental cows” following your suggestion (revised version, Line 145).

 

(25) Line 147 and 161: Please replace the day of the first GnRH treatment. The first GnRH should be administered on day 0 (in the 0-to-7-day description). See Fig. 1.

        We have revised the manuscript by adding an explanation of the first GnRH administration on day 0 (revised version, Lines 148-153 and 164-167).

 

(26) 2.3 Ultrasound assessment of follicular development and ovulation Comment: Terminal follicular development, or growth, is the growth phase that exhibit all gonadotropin-dependent follicles. From these cohorts the ovulatory follicles are selected for ovulation. Every follicle is involved in this TFG; therefore, it is not possible to refer to a terminal follicle (TF) to characterize an ovulatory wave. Please refer to recruited, selected and dominant follicles in this context. In addition, it is not clear what authors are referring to when they defined TF as the largest follicle (>7,0 mm) in the study (dominant or preovulatory follicles, but 7,0 mm and beyond?). Details of the ovulatory wave, the size of preovulatory follicles, and the interval CIDR removal to ovulation are also required (numbers are required).

        We have revised it following your suggestion in the revised manuscript (revised version, Lines 172-184).

 

(27) Line 173: It is better to describe the method of assessing follicular diameter just by mentioning the internal calipers that every US equipment has instead of longest and shortest diameters, because follicles are usually rounds.

        We have revised the method of assessing follicular diameter following your suggestion (revised version, Line 177).

 

(28) 2.4. Ultrasound assessment of CL diameter and CSA Lines 183-184: Please delete the statement. It is obvious.

        We have deleted the statement, following your suggestion, in the revised manuscript.

 

(29) Lines 184-185: Please replace “The developing CL was observed on days 5–6, and the mid-stage CL was identified between days 9–12 post-ovulation [33]” by CL development was study, (assessed/analyzed) in days 5-6 and 9-12 post ovulation, and select a reference related to the statement.

        Thank you for your suggestion. However, we need to indicate the day of CL evaluation, as we also noted that days 5-6 correspond to the developing CL and days 9-12 represent the mid-stage CL. Therefore, we used these stages to design the figure results (revised version, Lines 187-188).

 

(30) Line 189: Replace “cross-sectional area” by luteal area as it is the common name for this measure. It is required a statement that the luteal cavity was subtracted from the total area.

We have revised the manuscript following your suggestion, using luteal area instead of cross-sectional area. Additionally, we have included information to explain the luteal cavity, which was subtracted from the total area (revised version, Lines 186 and 191-194).

 

(31) Lines 191-193: Please delete the measure of volume as it does not add value to the luteal area to assess luteal development. In fact, it is rarely used.

        We have deleted it, following your suggestion, in the revised manuscript.

 

(32) Lines 196-197: This statement is not clear as it appears to consider luteal cavities as an abnormal feature. Please add the information obtained from all CLs.

        We have deleted the statement, as we have already included the information explaining the luteal cavity in the relevant section of the revised manuscript.

 

(33) 2.5. Analysis of Tissue and Colored Areas of the CL Lines 201-203: Please delete this statement. As above, it is understood.

        We have deleted it following your suggestion in the revised manuscript.

 

(34) 2.6. Assessment of Plasma P4 levels Lines 220-222: Again, please delete this statement and focus on P4 assessment.

        We have deleted it following your suggestion in the revised manuscript.

 

(35) Lines 230-234: Please delete this statement; any discussion to the right section.

        We have deleted it following your suggestion in the revised manuscript.

 

Comment: The ovulatory wave and ovulation need to be described in details to highlight the effect of eCG administered at day 4 on the patterns of follicular growth in this breed. Ovulations were registered and so should have been the diameters of ovulated follicles and the intervals between CIDR removal and ovulations in both groups. Again, to use this information in a more clinical settings, to compare to other studies in Bos indicus, to increase the precision in the description of results on follicular dynamics and ovulation and luteal dynamics and hemodynamics, authors are encouraged to describe it in Tables (means and deviations) and include these images to have representative information. The same is demanded, again, for describing the results from correlation analyses.

        Thank you for your valuable feedback. In this study, we did not present the values in the tables; however, we provided a scatter plot and explained the values in the text, including the standard error of the mean (SEM). The figure was used to visually represent the data, helping to convey the information more clearly. The correlations are also indicated, with asterisks marking significant values, and all correlation values are presented within the figure.

 

7. Discussion #2:

(36) Line 393: Not clear what authors refer by disrupting ovulation synchronization. Please precision is required.

        We have revised the statement for clarity (revised version, Lines 386-388).

 

(37) Line 403-406: Please delete this statement, it is very speculative. In fact, a recent review from Murphy (Animal Reproduction, 2012) stated that the effect of eCG still constitutes an unsolved question.

        We have deleted it following your suggestion in the revised manuscript.

 

(38) Lines 413-419: Please delete this paragraphs as those do not provide relevant support to the findings in this study.

        We have deleted it following your suggestion in the revised manuscript.

 

(39) Lines 425-428: Please delete this statement. The line of arguments should follows the statement immediately above. Authors need to remind that eCG was added at day 4 of CIDR treatment, and only 400 IU. As eCG has a long but limited biological activity, it could not directly affect the luteal development and function.

        Thank you for your comment. We have deleted it following your suggestion in the revised manuscript.

 

(40) Lines 433-436: Please delete this statement as it has not relationship to the study.

        We have deleted it following your suggestion in the revised manuscript.

 

(41) Lines 465-470: Please, again, delete this paragraph as there is not a temporal relationship between the eCG in this protocol with the CL development and function much later.

        We have deleted it following your suggestion in the revised manuscript.

 

Comment: The discussion was improved in terms of focus, but it still lacks in focus, precision, and conceptual framework. Authors should consider that eCG was added at day 4 in a 7-day CIDR protocol and in a very low dose. Therefore, the ECG influence on luteal development must be mediated by its impact on follicular development; indeed, the correlations found between follicular markers and luteal development bring support and should have been discussed with more precision. In addition, the terminology used to describe the follicular growth must be the conventional one, and the impact on the interval to ovulation was not considered.

        Thank you for your comment regarding the administration of eCG on day 4 of the 7-day CIDR protocol, and the concern about its low dose. In fact, eCG use in cattle is restricted, and the dosage is calculated based on the animal's weight prior to administration. Administering an overdose could lead to excessive superovulation, which is not beneficial for the animal. When eCG was administered during the estrous synchronization protocol in this study, we observed the development of a dominant follicle on the ovary, which responded to the eCG injection. Therefore, we did not focus extensively on the timing of eCG administration on day 4 in the estrous synchronization protocol. Instead, our primary focus was on demonstrating the benefits of eCG in stimulating follicular development, which in turn promotes CL development and P4 secretion. Specifically, we observed that larger CLs resulted in higher P4 production and increased CL blood perfusion.

 

8. Conclusions #2:

(41) Comment: The study showed that the addition of eCG to the ES protocol affected terminal follicular development, probably the ovulation interval and pattern and as a consequence on luteal development. Please review the conclusions, and the English writing, including that was either the eCG treatment that caused the effect or cows treated with eCG exhibited those effects.

        We have revised the conclusion section following your suggestion (revised version, Lines 455-467).

Author Response File: Author Response.docx