Innovation on Swine Semen Storage: Bacteriostatic Coating vs. Conventional Blister in Commercial Swine Semen Production

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript describes a study investigating the efficacy of a new antibacterial coating on blister packs for swine semen, to avoid the use of antibiotics. Such a study is important in the fight against antimicrobial resistance. Two studies were conducted, one to assess the effects of the antibacterial coating on bacterial load and sperm quality, and the second to investigate the effects on reproduction in an artificial insemination trial.  In general the manuscript is well-written, although there are some points that need to be addressed before the paper can be considered for publication. The major flaw seems to be that for the AI trial, the semen was extended in NutriXcell that already contains antibiotics. Therefore, the trial measures only the difference between a conventional blisterbag + antibiotics and the bactibag + antibiotics. If the main object is to find an alternative to antibiotics, why were antibiotics included? A trial including approximately 2000 sows testing two bags containing semen in antibiotics seems to be pointless – a similar effect could have been achieved with a small pilot study. A better test would be to compare the antibiotic-containing extender in the conventional bag and a non-antibiotic extender in the bactibag, especially since the in vitro analyses in Study 1 actually looked at such a comparison. Such a comparison would have real meaning in the fight against antimicrobial resistance.

There are several mentions of the One Conservation concept, which is the interconnection between ex situ and in situ conservation programs for rare and endangered species. This concept does not fit the subject under investigation. Do the authors mean the One Health concept – the interaction between animal health, human health and environmental health?

The use of the term kinetics is incorrect. Do the authors mean kinematics?

The paragraph in the Introduction on alternative methods to controlling bacterial multiplication with antibiotics is incomplete. Recent publications (several in the last 5 years) include separation of sperm from bacteria by physical means, which should be included here.

CASA analysis. At what temperature is the analysis conducted? It says that the samples were pre-warmed to 37°C. Is this the temperature of the analysis as well?

Bacteriology – what type of agar plates were used and what were the culture conditions? Why was incubation only carried out for 24h? Some bacteria in semen are slow -growing and would not be visible after only 24h.

Why was the semen quality analysis carried out on different ejaculates to the bacterial load? It would be interesting to see the effects of the bacteria on sperm quality, as in other studies published recently.

Sperm quality is much more than just motility and the absence of agglutination. Since some samples were retained at the lab for analysis, why were only these two properties evaluated on the stored samples?

Since semen was available from several breeds of boar, was boar breed included as a factor in the statistical analysis?  This would be an important issue of the Bactibag is to be used commercially.

The Results section contain several paragraphs that belong in the Discussion. According to the journal guidelines and template, the Results section should only only report results. Therefore, please move interpretation of the results to the Discussion. e.g. lines 272-277, and others

In Table 2, what is the unit µm/seg for VCL?

Author Response

We would like to begin by extending our sincere gratitude to the reviewers for their insightful and constructive feedback. We particularly appreciate the critical comments regarding the experimental design of the on-farm trial. In this regard, we wish to clarify that the primary intention of this large-scale experiment was to evaluate the impact and safety of the bacteriostatic blister on semen quality and pregnancy rates under existing commercial conditions. The aim was not to re-validate the blister's efficacy in bacterial control, as this property has already been extensively proven and is detailed in the product's patent. Our goal was to confirm that this new material did not negatively affect fertility, a crucial first step before advocating for subsequent trials without antibiotics in a commercial setting. We have revised the manuscript to make this objective clearer.

Comments 1: The manuscript describes a study investigating the efficacy of a new antibacterial coating on blister packs for swine semen, to avoid the use of antibiotics. Such a study is important in the fight against antimicrobial resistance. Two studies were conducted, one to assess the effects of the antibacterial coating on bacterial load and sperm quality, and the second to investigate the effects on reproduction in an artificial insemination trial.  In general the manuscript is well-written, although there are some points that need to be addressed before the paper can be considered for publication.

Response 1: We sincerely thank the reviewer for their thorough evaluation and constructive feedback, which have significantly helped improve the quality of our manuscript. Below, we address each of the points raised.

Comments 2: The major flaw seems to be that for the AI trial, the semen was extended in NutriXcell that already contains antibiotics. Therefore, the trial measures only the difference between a conventional blisterbag + antibiotics and the bactibag + antibiotics. If the main object is to find an alternative to antibiotics, why were antibiotics included? A trial including approximately 2000 sows testing two bags containing semen in antibiotics seems to be pointless – a similar effect could have been achieved with a small pilot study. A better test would be to compare the antibiotic-containing extender in the conventional bag and a non-antibiotic extender in the bactibag, especially since the in vitro analyses in Study 1 actually looked at such a comparison. Such a comparison would have real meaning in the fight against antimicrobial resistance.

Response 2: We sincerely appreciate this critical observation regarding the inclusion of antibiotics in the extender used for the commercial trial. We agree that a direct comparison between conventional bags with antibiotic-containing extender and BactiBag® with antibiotic-free extender would represent an ideal scenario for evaluating antibiotic replacement. However, the primary objective of this large-scale field trial was to validate the safety, compatibility, and efficacy of the new blister material under existing, real-world industry conditions where the use of antibiotics in semen extenders remains standard practice. Our first step was to demonstrate that the novel bag performs at least as well as conventional packaging within this established system, without negatively impacting fertility. This initial validation was crucial for gaining industry acceptance before proceeding to subsequent trials with antibiotic-free extenders. We have clarified this rationale in the Discussion section, emphasizing that future studies will explore the antibiotic-free comparison.

Comments 3: There are several mentions of the One Conservation concept, which is the interconnection between ex situ and in situ conservation programs for rare and endangered species. This concept does not fit the subject under investigation. Do the authors mean the One Health concept – the interaction between animal health, human health and environmental health?

Response 3: We sincerely thank the reviewer for pointing out this error. The reviewer is correct; we intended to refer to the One Health concept. We have corrected this term throughout the manuscript.

Comments 4: The use of the term kinetics is incorrect. Do the authors mean kinematics?

Response 4: Thank you for this correction. We agree that 'kinematics' is the precise term to describe sperm motion characteristics, and we have amended the text accordingly throughout the manuscript.

Comments 5: The paragraph in the Introduction on alternative methods to controlling bacterial multiplication with antibiotics is incomplete. Recent publications (several in the last 5 years) include separation of sperm from bacteria by physical means, which should be included here.

Response 5: We appreciate the suggestion to expand on alternative methods in the Introduction. We agree that strengthening this section provides better context for our study. We have updated the Introduction to include broader strategies for bacterial control beyond antibiotics. The Introduction (Section 1, fifth paragraph) now includes: "Research has explored alternative strategies to reduce bacterial contamination and proliferation in semen without relying on antibiotics. These include improving hygiene during semen collection, processing, and storage, as well as using antimicrobial supplements in AI doses or storing them at lower temperatures."

Comments 6: CASA analysis. At what temperature is the analysis conducted? It says that the samples were pre-warmed to 37°C. Is this the temperature of the analysis as well?

Response 6:  Thank you for requesting this clarification. We confirm that the CASA analysis was indeed performed at 37°C, the same temperature as the pre-incubation. We have clarified this detail in the Methods section to ensure full transparency of our protocol.

Comments 7: Bacteriology – what type of agar plates were used and what were the culture conditions? Why was incubation only carried out for 24h? Some bacteria in semen are slow -growing and would not be visible after only 24h.

Response 7: We apologize for this oversight in the original submission. We agree that detailed information on bacteriological methods is crucial for reproducibility. We have corrected the Methods section to specify that cultures were performed on Tryptic Soy Agar (TSA) plates and incubated for 48 hours, which, as the reviewer rightly pointed out, allows for the detection of slower-growing bacteria.

Comments 8: Why was the semen quality analysis carried out on different ejaculates to the bacterial load? It would be interesting to see the effects of the bacteria on sperm quality, as in other studies published recently.

Response 8: We appreciate the opportunity to clarify this point. The sperm quality analyses were indeed performed on a subset of the same ejaculates used for the bacteriology assessment. We have revised the Methods section to explicitly state this, ensuring the direct link between these two analyses is clear.

Comments 9: Sperm quality is much more than just motility and the absence of agglutination. Since some samples were retained at the lab for analysis, why were only these two properties evaluated on the stored samples?

Response 9: Thank you for this valuable comment. We agree that a comprehensive assessment of sperm quality is essential. We would like to clarify that in addition to total and progressive motility and agglutination, our study already included a detailed kinematic evaluation using the CASA system. The parameters measured and presented in the manuscript are ALH, BCF, VCL, VAP, VSL, STR, and LIN, providing a robust analysis of sperm functionality.

Comments 10: Since semen was available from several breeds of boar, was boar breed included as a factor in the statistical analysis?  This would be an important issue of the Bactibag is to be used commercially.

Response 10: We greatly appreciate this important question. We confirm that boar breed was indeed included as a factor in our statistical analysis for all assessed sperm quality parameters. We found no significant interaction between breed and the blister type, a finding highly relevant for the commercial application of the BactiBag® as it suggests consistent effectiveness across various boar breeds. This point has been emphasized in the manuscript.

Comments 11: The Results section contain several paragraphs that belong in the Discussion. According to the journal guidelines and template, the Results section should only only report results. Therefore, please move interpretation of the results to the Discussion. e.g. lines 272-277, and others

Response 11: We agree with the reviewer's guidance on adhering to journal structure. We have carefully reviewed the manuscript and moved all interpretive paragraphs from the Results section to the Discussion section, ensuring that the Results section now solely presents the direct findings of our study.

Comments 12: In Table 2, what is the unit µm/seg for VCL?

Response 12: Thank you for catching this typographical error. We agree, and the unit has been corrected to 'µm/s' in Table 2.

Reviewer 2 Report

Comments and Suggestions for Authors

Line 87 – Please add what device did you use to pre-incubate processed semen samples before CASA assessment.

Line 101 - Please write which commercial pig hybrids were used in the experiment, their names.

Line 103 - Is this age of the animal important to you because of the biological test or because animals at this age are considered sexually mature?

Line 109 – after words using the double-gloved hand technique, I will add: to provide a double layer of protection from contamination.

Line 113 – If it possible can you write what is the number of rejected samples that do not satisfy these criteria?

Line 150 – you have double word were (were were stored), maybe you meant on where were stored?

Line 207 – Please explain in one sentence did the female were kept in individual boxes after insemination for example 21 days (because of possibility a failure to conceive of a sows or gilts when they kept in groups in the first days after insemination).

Line 216 - What method did you use to determine estrus in sows and gilts? Pleas add that information.

Line 219 - Please specify which Ultrasound machine have you used?

Line 259-261 – What do you think, what is the reason of it?

Line 388 – You can add: This affects the economics of pig production and the rational use of boars.

Line 529 - Have you examined the occurrence of light piglets, avital piglets, mummified piglets and stillborn piglets? If so, in which treatment were the best litters?

Author Response

We would like to begin by extending our sincere gratitude to the reviewers for their insightful and constructive feedback. We particularly appreciate the critical comments regarding the experimental design of the on-farm trial. In this regard, we wish to clarify that the primary intention of this large-scale experiment was to evaluate the impact and safety of the bacteriostatic blister on semen quality and pregnancy rates under existing commercial conditions. The aim was not to re-validate the blister's efficacy in bacterial control, as this property has already been extensively proven and is detailed in the product's patent. Our goal was to confirm that this new material did not negatively affect fertility, a crucial first step before advocating for subsequent trials without antibiotics in a commercial setting. We have revised the manuscript to make this objective clearer.

Comments 1: Line 87 – Please add what device did you use to pre-incubate processed semen samples before CASA assessment.

Response 1: We appreciate the request for further detail regarding the pre-incubation device. While the specific equipment was not explicitly noted in the experimental protocol, samples were pre-incubated at 37°C for 10 minutes to ensure thermal equilibration. This procedure is typically carried out using a temperature-controlled dry bath to maintain consistent conditions.

Comments 2: Line 101 - Please write which commercial pig hybrids were used in the experiment, their names.

Response 2: Thank you for this pertinent request for additional detail. We have added the names of the commercial hybrids used in the experiment, specifying them as Landrace x Large White, to enhance the completeness of our methods description.

Comments 3: Line 103 - Is this age of the animal important to you because of the biological test or because animals at this age are considered sexually mature?

Response 3:  We appreciate your question regarding the age of the animals. The average age of 13.3 ± 3.8 months was important as it ensures the males are considered sexually mature with established fertility profiles, thus ensuring the biological relevance and validity of our tests. This detail has been clarified in the manuscript.

Comments 4: Line 109 – after words using the double-gloved hand technique, I will add: to provide a double layer of protection from contamination.

Response 4: Thank you for this suggestion. We agree that adding this detail enhances the clarity of our methodology. The requested information has been incorporated into the manuscript.

Comments 5: Line 113 – If it possible can you write what is the number of rejected samples that do not satisfy these criteria?

Response 5: We appreciate this inquiry regarding the number of rejected samples. While the exact count of rejected ejaculates was not recorded for this specific study, we can confirm that rigorous selection criteria were applied: only normospermic ejaculates with at least 70% motility and a maximum of 20% abnormal sperm cells (according to CASA results) were utilized. We agree on the importance of this information for complete experimental description and will endeavor to include such data in future studies.

Comments 6: Line 150 – you have double word were (were were stored), maybe you meant on where were stored?

Response 6: Thank you for pointing out this typographical error. The grammatical correction has been made in the manuscript.

Comments 7: Line 207 – Please explain in one sentence did the female were kept in individual boxes after insemination for example 21 days (because of possibility a failure to conceive of a sows or gilts when they kept in groups in the first days after insemination).

Response 7: We appreciate the request for more detailed information on female housing. To ensure optimal conditions for conception, the females (sows and gilts) were kept in individual pens during the AI. They remained in these stalls until pregnancy was confirmed via ultrasonography, 30 days post-insemination. Only after this confirmation were the pregnant females transferred to group pens. This protocol is now clearly described in the manuscript.

Comments 8: Line 216 - What method did you use to determine estrus in sows and gilts? Pleas add that information.

Response 8: Thank you for highlighting this missing detail. We have added the specific method for estrus detection in sows and gilts, which involved performing the back-pressure test (BPT) twice daily in the presence of a boar, to the manuscript for clarity.

Comments 9: Line 219 - Please specify which Ultrasound machine have you used?

Response 9: We appreciate the request for precision regarding the equipment used. We have specified the ultrasound machine as 'SC Tecnoscan, IMV Imaging, Bellshill, UK' in the relevant section of the manuscript.

Comments 10: Line 259-261 – What do you think, what is the reason of it?

Response 10: We appreciate your insightful question. The underlying reasons for the observed differences in sperm kinetic profiles are discussed in detail in the Discussion section of the manuscript. Our hypothesis centers on the bacteriostatic blister's ability to limit bacterial proliferation and endotoxin release, thereby better preserving sperm structural integrity and energetic capacity, which leads to a more vigorous swimming pattern.

Comments 11: Line 388 – You can add: This affects the economics of pig production and the rational use of boars.

Response 11: Thank you for this excellent suggestion. We agree that this phrase effectively highlights the broader impact of semen preservation. We have incorporated it into the Discussion section to strengthen the context of our study.

Comments 12: Line 529 - Have you examined the occurrence of light piglets, avital piglets, mummified piglets and stillborn piglets? If so, in which treatment were the best litters?

Response 12: We appreciate this valuable suggestion for a more comprehensive assessment of reproductive outcomes. While the occurrence of light, avital, mummified, or stillborn piglets was not within the scope of the parameters evaluated in this specific study, as the experiment was performed in a commercial farm, we acknowledge their importance for a complete understanding of reproductive performance. We consider this a highly relevant point and plan to include such evaluations in future research under controlled experimental conditions (non-commercial settings).

Reviewer 3 Report

Comments and Suggestions for Authors

Regarding the manuscript entitled “Innovation on Swine Semen Storage: Bacteriostatic Coating vs. Conventional Blister in Commercial Swine Semen Production”, I consider that the study requires major revision before it can be considered for publication. Although the topic addressed—the potential replacement of antibiotics in boar semen doses using a bacteriostatic-coated blister—is relevant, the current experimental design does not support solid conclusions regarding its efficacy, and the claimed novelty of the work is not sufficiently justified.

One of the main concerns is that the experiments are not designed to demonstrate the bacteriostatic effect of the coating. To assess such an effect, it is essential to conduct in vitro assays with controlled bacterial contamination and to test semen doses without antibiotics. However:

• No trials were conducted using deliberately contaminated semen, which prevents any evaluation of the coating’s ability to control bacterial growth. The ejaculates used had very low or even negligible bacterial loads, as acknowledged by the authors.
• All semen doses included antibiotics, so any possible effect of the coating cannot be separated from the action of the extender. This seriously undermines any conclusion regarding the blister’s capacity to replace antibiotics.
• The concentration of the active compound in the coating is neither reported nor discussed, nor is its consistency across batches. This omission affects the reproducibility and scientific validity of the study.
• There is no proper control group (i.e., uncoated blister + semen without antibiotics) to allow evaluation of the specific effect of the coating. This lack of critical controls severely limits the interpretation of results.

Overall, the experimental design only allows the authors to confirm the safety of the coated material in terms of semen quality and reproductive performance, but not its antimicrobial capacity or its suitability as an alternative to antibiotics.

Furthermore, the coated blister being tested has already been promoted by the manufacturing company (IMV Technologies), with in vitro and on-farm data publicly available since at least 2014 (as included in the leaflet attached by the authors). Additionally, a study by Camugli et al. (2019), cited as reference #39, already reported on-farm trials using this blister without antibiotics. In this context, it is essential that the authors clearly explain what the current study adds to the existing knowledge. What is the novel hypothesis? What new evidence is being provided?

Finally, the manuscript advocates for the use of this blister as a more sustainable solution without critically discussing the potential risks associated with triclosan, the active compound in the coating. This substance has been subject to restrictions in several countries due to its potential as an endocrine disruptor. Therefore, its use should be properly justified and discussed from both a safety and environmental perspective.

Below are the specific comments that should be addressed individually and thoroughly in the revised version.

SPECIFIC COMMENTS

1. Study objective and experimental design (Lines 16–17 and 61–64)

• The stated objective is to evaluate the bacteriostatic effect of the coated blister. However, the study lacks the appropriate tests to assess such an effect.
• The authors should justify why no in vitro contamination trials were performed using the actual concentration of the active compound.
• It is also necessary to explain why semen without antibiotics was not included, which would be essential to determine the blister’s independent antimicrobial effect.

2. Initial contamination level (Line 99)

• The initial bacterial contamination of raw semen is not reported. Table 1 suggests it was very low or absent, making it impossible to assess the antimicrobial performance of the coating.
• The authors should clarify whether microbiological analyses were performed before packaging, and what methods were used. If not, this limitation should be explicitly acknowledged.

3. On-farm trial design and timing (Lines 168 and 203)

• All doses used in the field trial contained antibiotics, making it impossible to determine any antimicrobial contribution from the blister.
• Moreover, microbiological analysis was conducted at 72 and 120 hours, whereas inseminations occurred between 24 and 48 hours.
• The authors should justify this mismatch in sampling time and acknowledge that no direct relationship can be drawn between microbial load and reproductive outcome.

4. Microbiological results and controls (Lines 254, 262–277)

• A proper negative control (uncoated blister + semen without antibiotics) was not included, which is critical to interpret any bacteriostatic effect.
• According to the results, the CB–A group (coated blister without antibiotics) showed a significant bacterial increase after 120 h, comparable to the control group.
• The authors should explain how they conclude that “bacterial control” occurred when no significant reduction was observed.

5. Interpretation of reproductive results (Lines 296–299 and 366–371)

• The reproductive performance data merely confirm that the blister is not harmful, but do not support any benefit.
• The authors should reformulate their conclusions to reflect this and acknowledge that their design does not allow testing of the antimicrobial hypothesis.

6. Discussion: unsupported hypothesis (Lines 403–443)

• Although the working hypothesis is sound, the study does not provide the necessary evidence to test it.
• No information is given regarding the concentration of the coating compound or its antimicrobial efficacy against common bacteria.
• The discussion should be revised accordingly, recognizing the methodological limitations and the absence of direct validation.

7. Triclosan use: safety and sustainability (Line 546)

• The manuscript claims this approach aligns with sustainable practices, but the safety and regulatory context of triclosan is not discussed.
• The authors should specifically address the toxicological, environmental, and regulatory concerns of using triclosan in materials that come into contact with biological products, and explain how the blister is managed post-use to avoid environmental contamination.

 

Comments for author File: Comments.pdf

Author Response

We would like to begin by extending our sincere gratitude to the reviewers for their insightful and constructive feedback. We particularly appreciate the critical comments regarding the experimental design of the on-farm trial. In this regard, we wish to clarify that the primary intention of this large-scale experiment was to evaluate the impact and safety of the bacteriostatic blister on semen quality and pregnancy rates under existing commercial conditions. The aim was not to re-validate the blister's efficacy in bacterial control, as this property has already been extensively proven and is detailed in the product's patent. Our goal was to confirm that this new material did not negatively affect fertility, a crucial first step before advocating for subsequent trials without antibiotics in a commercial setting. We have revised the manuscript to make this objective clearer.

Comments 1: Regarding the manuscript entitled “Innovation on Swine Semen Storage: Bacteriostatic Coating vs. Conventional Blister in Commercial Swine Semen Production”, I consider that the study requires major revision before it can be considered for publication. Although the topic addressed—the potential replacement of antibiotics in boar semen doses using a bacteriostatic-coated blister—is relevant, the current experimental design does not support solid conclusions regarding its efficacy, and the claimed novelty of the work is not sufficiently justified.

One of the main concerns is that the experiments are not designed to demonstrate the bacteriostatic effect of the coating. To assess such an effect, it is essential to conduct in vitro assays with controlled bacterial contamination and to test semen doses without antibiotics. However:

• No trials were conducted using deliberately contaminated semen, which prevents any evaluation of the coating’s ability to control bacterial growth. The ejaculates used had very low or even negligible bacterial loads, as acknowledged by the authors.
• All semen doses included antibiotics, so any possible effect of the coating cannot be separated from the action of the extender. This seriously undermines any conclusion regarding the blister’s capacity to replace antibiotics.
• The concentration of the active compound in the coating is neither reported nor discussed, nor is its consistency across batches. This omission affects the reproducibility and scientific validity of the study.
• There is no proper control group (i.e., uncoated blister + semen without antibiotics) to allow evaluation of the specific effect of the coating. This lack of critical controls severely limits the interpretation of results.

Overall, the experimental design only allows the authors to confirm the safety of the coated material in terms of semen quality and reproductive performance, but not its antimicrobial capacity or its suitability as an alternative to antibiotics.

Response 1: We sincerely thank the reviewer for this critical and well-founded observation. We entirely agree that the experimental design, particularly the on-farm trial, does not allow for a direct conclusion about the blister's ability to replace antibiotics. Our primary objective for this study was different, and we apologize that this was not made clear in the original manuscript. The goal was to validate the safety and non-inferiority of the new bacteriostatic material compared to the conventional blister under standard commercial conditions (which include antibiotics). This was a crucial first step to ensure the material had no detrimental effects on sperm quality or fertility before proceeding with more advanced, antibiotic-free trials. We have thoroughly revised the Introduction, Objectives, and Discussion sections to correctly frame the study's scope around this safety and performance validation.

Comments 2: Furthermore, the coated blister being tested has already been promoted by the manufacturing company (IMV Technologies), with in vitro and on-farm data publicly available since at least 2014 (as included in the leaflet attached by the authors). Additionally, a study by Camugli et al. (2019), cited as reference #39, already reported on-farm trials using this blister without antibiotics. In this context, it is essential that the authors clearly explain what the current study adds to the existing knowledge. What is the novel hypothesis? What new evidence is being provided?

Response 2: The reviewer raises a very important point regarding the novelty and contribution of our work. The work presented at the Proceedings of the IX International Conference of Boar Semen Preservation by Camugli et al. (2019) was only a congress abstract, which typically presents preliminary data and lacks the detailed methodology and peer-reviewed rigor of a full scientific paper. Furthermore, our study provides a substantial advancement in several key areas: (1) Scale: Our field trial is significantly larger (2,192 females vs. approx. 384), providing much greater statistical power and reliability. (2) Insemination Technique: Our trial included a large proportion of post-cervical inseminations, a modern technique not specified in the abstract. (3) Environmental Conditions: Our study was conducted in a tropical climate, which presents greater challenges for semen preservation compared to the temperate European climate where the previous trial was likely conducted. Therefore, our manuscript represents the first comprehensive, large-scale, peer-reviewed validation of this technology's impact on semen quality and fertility under challenging real-world conditions.

Furthermore, regarding the promotional material referenced by the reviewer, a closer examination reveals that it does not constitute peer-reviewed scientific evidence. On page one, the data presented is from the Camugli et al. conference abstract, not a full, peer-reviewed article. The scientific articles referenced on page three are not related to the BactiBag® technology itself. The data shown on page four consists of internal company findings, which are presented without the rigorous statistical analysis necessary for independent scientific validation or to draw firm conclusions. Therefore, the information within these materials lacks the solid scientific foundation that only a peer-reviewed publication can provide. Our study was conceived precisely to fill this gap. In technically demanding markets, such as Brazil, product claims require robust, scientifically-backed evidence. Our research was designed to provide this necessary level of validation, ensuring the findings are transparent, reproducible, and critically evaluated by the scientific community.

Comments 3: Finally, the manuscript advocates for the use of this blister as a more sustainable solution without critically discussing the potential risks associated with triclosan, the active compound in the coating. This substance has been subject to restrictions in several countries due to its potential as an endocrine disruptor. Therefore, its use should be properly justified and discussed from both a safety and environmental perspective.

Response 3: We are very grateful to the reviewer for raising this critical point. It was a significant oversight in our original manuscript. We agree that a discussion of sustainability must be balanced and address the full lifecycle of the product. We have now added a detailed paragraph to the Discussion section addressing the environmental and regulatory context of using Triclosan. This new section clarifies that the post-use blister is classified as Class I Hazardous Waste (in Brazil and UE) while it is not in the USA, and details the legally mandated disposal protocols, including segregation and collection by licensed contractors for incineration or disposal in a specialized landfill. This provides a much more complete and responsible assessment of the technology.

 

Below are the specific comments that should be addressed individually and thoroughly in the revised version.

SPECIFIC COMMENTS

Comments 4: Study objective and experimental design (Lines 16–17 and 61–64)

• The stated objective is to evaluate the bacteriostatic effect of the coated blister. However, the study lacks the appropriate tests to assess such an effect.
• The authors should justify why no in vitro contamination trials were performed using the actual concentration of the active compound.
• It is also necessary to explain why semen without antibiotics was not included, which would be essential to determine the blister’s independent antimicrobial effect.

Response 4: We appreciate the reviewer's attention to the clarity of our stated objectives. We acknowledge that the phrasing in the original submission might have been imprecise. Our experiment was designed to primarily evaluate the efficacy of the bacteriostatic-coated blister in preserving sperm quality during storage and its impact on reproductive performance in a commercial setting. While the underlying bacteriostatic properties of the product are foundational to its design and have been previously detailed in its patent, the core of this study was to demonstrate its practical utility in maintaining semen quality and fertility. We have refined the language in the abstract and introduction to reflect this primary focus on semen quality preservation and reproductive outcomes more accurately, while also maintaining transparency regarding the product's inherent bacteriostatic nature.

 

Comments 5: Initial contamination level (Line 99)

• The initial bacterial contamination of raw semen is not reported. Table 1 suggests it was very low or absent, making it impossible to assess the antimicrobial performance of the coating.
• The authors should clarify whether microbiological analyses were performed before packaging, and what methods were used. If not, this limitation should be explicitly acknowledged.

Responde 5: We acknowledge the importance of measuring baseline bacterial contamination for a complete understanding of antimicrobial performance. While a formal quantification of initial bacterial load in raw semen was not performed at day 0 in this specific experiment, our focus was on bacterial proliferation within the stored semen doses over time under different conditions. We agree that including initial contamination data would further strengthen the study's microbial assessment and will consider this for future research designs.

 

Comments 6: On-farm trial design and timing (Lines 168 and 203)

• All doses used in the field trial contained antibiotics, making it impossible to determine any antimicrobial contribution from the blister.
• Moreover, microbiological analysis was conducted at 72 and 120 hours, whereas inseminations occurred between 24 and 48 hours.
• The authors should justify this mismatch in sampling time and acknowledge that no direct relationship can be drawn between microbial load and reproductive outcome.

Responde 6: We agree with the reviewer's observation regarding the timing of our microbiological assessments relative to the commercial insemination window. The sampling points for bacterial proliferation (72 and 120 hours) were chosen based on previous research indicating the typical onset of significant bacterial growth in swine semen doses. The 24–48 hour insemination schedule, conversely, reflects standard commercial farm practice where the experiment was conducted. While this means a direct, real-time correlation between the highest bacterial loads and reproductive outcomes cannot be definitively established from this specific trial, our objective was to evaluate semen quality and reproductive performance under conditions reflective of commercial operations. We acknowledge this aspect of the experimental design and its implications for interpreting the microbiological data within the context of immediate field application.

 

Comments 7: Microbiological results and controls (Lines 254, 262–277)

• A proper negative control (uncoated blister + semen without antibiotics) was not included, which is critical to interpret any bacteriostatic effect.
• According to the results, the CB–A group (coated blister without antibiotics) showed a significant bacterial increase after 120 h, comparable to the control group.
• The authors should explain how they conclude that “bacterial control” occurred when no significant reduction was observed.

Response 7: We appreciate the reviewer's scrutiny of our microbiological controls and interpretation. We would like to clarify that a negative control group (conventional blister without antibiotic, labeled as 'CB-A') was included in our experimental design, as detailed in Section 2.2 and presented in Table 1. Our analysis indicated that at 72 hours, no significant differences in bacterial growth were observed among the bacteriostatic coating blister with antibiotics, bacteriostatic coating blister without antibiotics, and the Control with antibiotics groups. While bacterial growth in the 'bacteriostatic coating blister without antibiotic' group was higher at 120 hours compared to antibiotic-containing groups, the overall bacterial contamination remained generally low, with most measurements below 2 log CFU/mL, indicating a baseline level of control provided by the bacteriostatic coating itself, even in the absence of additional antibiotics in the extender.

 

Comments 8: Interpretation of reproductive results (Lines 296–299 and 366–371)

• The reproductive performance data merely confirm that the blister is not harmful, but do not support any benefit.
• The authors should reformulate their conclusions to reflect this and acknowledge that their design does not allow testing of the antimicrobial hypothesis.

Response 8: We agree with the reviewer's observation that our reproductive performance data primarily demonstrate that the bacteriostatic blister is not detrimental to fertility, maintaining results comparable to conventional methods. Indeed, a key objective of this study, especially given its commercial field setting, was to confirm that the new technology does not negatively impact established reproductive benchmarks. We have ensured that our conclusions reflect this finding, highlighting the blister as a viable and safe alternative or complement to existing approaches.

 

Comments 9: Discussion: unsupported hypothesis (Lines 403–443)

• Although the working hypothesis is sound, the study does not provide the necessary evidence to test it.
• No information is given regarding the concentration of the coating compound or its antimicrobial efficacy against common bacteria.
• The discussion should be revised accordingly, recognizing the methodological limitations and the absence of direct validation.

Response 9: We appreciate the reviewer's concern regarding the foundational evidence for our hypothesis. We agree that this needed further clarification. Our study's hypothesis is indeed built upon the pre-existing and validated technological foundation of the BactiBag® blister, whose bacteriostatic properties are detailed in its patent documentation. We have revised the Discussion section to include specific technical details from the patent, including the active agent concentration range (1 mg/m² to 40 mg/m²), as well as references to the in-vitro studies that demonstrate its efficacy in controlling bacterial proliferation. This inclusion supports the rationale for our study, which focused on evaluating its impact on sperm quality and reproductive performance under large-scale commercial conditions, rather than re-validating the core bacteriostatic mechanism.

 

Comments 10: Triclosan use: safety and sustainability (Line 546)

• The manuscript claims this approach aligns with sustainable practices, but the safety and regulatory context of triclosan is not discussed.
• The authors should specifically address the toxicological, environmental, and regulatory concerns of using triclosan in materials that come into contact with biological products, and explain how the blister is managed post-use to avoid environmental contamination.

Response 10:  We thank the reviewer for this important observation. You are correct that the original manuscript lacked the necessary background to support the working hypothesis. We have substantially revised the Discussion section to address this. It now explicitly states that due to the active antimicrobial agent, the post-use blister must be classified as hazardous waste according to European Union and Brazil regulations, while in the United States, the federal regulatory framework, it results in a non-hazardous waste classification. We detail the mandatory rigorous waste management practices required, including segregation, collection by licensed companies, and specialized disposal methods such as incineration or Class I hazardous waste landfill. This addition provides a more complete and responsible assessment of the technology, acknowledging that its benefits are contingent upon proper environmental management.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have improved the paper and addressed some of my comments. However, the Introduction is still incomplete since several publications have described AI trials in which separation of boar sperm from bacteria using a colloid was performed. These studies should be mentioned to provide a complete picture of alternatives. It would be interesting to note, for example, whether the economics of the two techniques were comparable, given the necessity to manufacture the special coating for the blister bags and to dispose of the bags after use.

There are many other analyses of sperm quality than can be carried out apart from CASA and agglutination, such as flow cytometry to evaluate membrane integrity, chromatin integrity, mitochondrial membrane potential, Reactive Oxygen Species production, ATP production, metabolic profiles etc. It is arguable that CASA  analysis provides only a very narrow indication of sperm quality. Why did you only  focus on sperm kinematics and agglutination?

In Tables 6 and 7, you refer to total number of piglets born and total live-born. However, these figures look as if they are per litter, not the overall total, whereas the other variables measured refer to all the sows. Please clarify.

Author Response

Dear Reviewer,

We would like to take this opportunity to clarify a central point regarding the study's scope. The primary objective of this experiment was to evaluate the impact of the bacteriostatic blister on the maintenance of semen quality and its subsequent effect on in vivo pregnancy rates under commercial conditions. The material's efficacy in bacterial control has already been extensively validated during the product's patenting process and, therefore, was not the focus of the investigation presented in this manuscript. Our goal was to assess its performance and safety in a real-world production environment

[Comment 1] The authors have improved the paper and addressed some of my comments. However, the Introduction is still incomplete since several publications have described AI trials in which separation of boar sperm from bacteria using a colloid was performed. These studies should be mentioned to provide a complete picture of alternatives. It would be interesting to note, for example, whether the economics of the two techniques were comparable, given the necessity to manufacture the special coating for the blister bags and to dispose of the bags after use.

[Response 1]
We thank the reviewer for this insightful recommendation. We agree that a discussion of colloid-based sperm separation is essential for providing a complete overview of antibiotic-free alternatives.

The manuscript has been substantially revised to address this point. We have now included this context in the Introduction and, more significantly, added a detailed paragraph to the Discussion section. This new section directly addresses the reviewer's suggestion by providing an economic comparison between the colloid centrifugation technique and the BactiBag system, thereby offering a more balanced perspective on the practical and financial viability of available technologies.

[Comment 2] There are many other analyses of sperm quality than can be carried out apart from CASA and agglutination, such as flow cytometry to evaluate membrane integrity, chromatin integrity, mitochondrial membrane potential, Reactive Oxygen Species production, ATP production, metabolic profiles etc. It is arguable that CASA  analysis provides only a very narrow indication of sperm quality. Why did you only focus on sperm kinematics and agglutination?

[Response 2]
The reviewer raises a very important point regarding the depth of sperm quality analysis. We agree that advanced techniques such as flow cytometry provide a more detailed biological assessment of sperm health.

However, the primary objective of this study was to evaluate the practical impact of the bacteriostatic blister under large-scale commercial conditions, with a focus on parameters directly correlated with in vivo fertility outcomes. Therefore, we selected CASA and agglutination analysis, as these are standard, industry-relevant methods that allow for a robust assessment of sperm functionality in a field setting. This methodological choice aligns with similar large-scale field studies, such as Basioura et al. (2025), which also relied on these parameters to link semen quality to fertility.

While a deeper molecular analysis is certainly valuable, it fell outside the scope of this particular applied research. We acknowledge this as a valuable direction for future laboratory-based investigations.

[Comment 3] In Tables 6 and 7, you refer to total number of piglets born and total live-born. However, these figures look as if they are per litter, not the overall total, whereas the other variables measured refer to all the sows. Please clarify.

[Response 3]
We thank the reviewer for identifying this ambiguity and helping us improve the clarity of our data presentation. The headers in Tables 6 and 7 were indeed potentially misleading.

We have revised them to explicitly state "Average Number of Piglets Born per Litter" and "Average Number of Live-Born Piglets per Litter" to ensure the data are correctly interpreted as per-litter averages.

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have addressed most of my comments satisfactorily, providing a more balanced comparison of the new Bactibag with other available technologies. However, as they themselves mention, sperm viability and metabolism are important for fertility yet neither membrane integrity nor mitochondrial membrane potential were evaluated. I would add cjhroamtin integirty as being particuarly important, especially where sperm doses are to be stored for several days. Instead, the authors claim to have focussed on parameters directly associated with fertility outcomes (kinematics and agglutination). This contradiction should be addressed e.g. by pointing out the limitations of this study. The authors should consider a more thorough evaluation of sperm quality in future studies.

Author Response

Thank you very much for your insightful considerations and constructive comments, which have certainly contributed to the improvement of our manuscript.

We have accepted your suggestion and inserted a new paragraph in the Discussion section to address the limitations of the in vitro assessment and to reinforce the need for more in-depth analyses in future studies. Bellow, the content included:

 

This study primarily focused on sperm quality parameters directly associated with field fertility outcomes, specifically kinematics and agglutination. However, a more comprehensive evaluation of sperm physiology was not performed. Analyzing attributes such as plasma and acrosomal membrane integrity, mitochondrial membrane potential, and chromatin integrity would offer a more complete and mechanistic understanding of the bacteriostatic blister's effects, particularly during prolonged storage periods.
While CASA and the assessment of conception and birth rates provide objective and predictive data highly relevant to commercial settings, they represent only one facet of complex sperm viability. Consequently, the absence of a more detailed physiological analysis is acknowledged as a methodological limitation of this study.
Future research should consider incorporating advanced techniques, such as flow cytometry, to investigate these subcellular parameters. Such an in-depth approach would complement the robust reproductive performance results presented herein, simultaneously validating the technology's safety and impact at a more detailed physiological level.