Review Reports - Ionic Gelation for Nano-Delivery of Sulforaphane in Animal Feed: A Conceptual Review of Stability, Efficacy, and Translation Potential

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The review conflates general SFN nanoencapsulation literature (mostly from human pharmaceutical and food science contexts) with the stated focus on animal feed, which is actually covered by very few of the cited studies.
The authors explicitly acknowledge on page 8 (lines 255–257): "there is a lack of precise studies that capitalize on encapsulating SFN using IG for animal feed" — which fundamentally undermines the review's premise and scope.
The review adds limited conceptual novelty beyond what existing SFN microencapsulation reviews already cover.
Heading numbering is erratic and inconsistent throughout. Section 1 ("Introduction") is followed by another "1." (Ionic gelation as an engineering framework) then another “1” (Engineering stability: encapsulation design and protective performance). Then later sections appear as "1." again (Measurable efficacy in animal models, p. 10). This indicates the manuscript was assembled from separate components without proper integration.
Sub-section numbering is mismatched with parent sections. For example, sub-sections labeled 2.2 and 2.3 appear before section 2 is introduced; section 3.1 appears before 3 is formally opened.
The abstract mentions chitosan and alginate as the primary focus, but the body expands broadly into liposomes, silk fibroin nanoparticles, PLGA, PEGylated nanoparticles, and dendrimers — far beyond ionic gelation.
Table 1 is titled "Gefitinib nanocarrier comparison" — Gefitinib is an anticancer drug with no relevance to sulforaphane or animal feed.
Several paragraphs (particularly in Section 3.4, pp. 9–10) discuss CpG oligonucleotides, immunotherapy, and cancer vaccine applications — topics entirely unrelated to the manuscript's stated scope.
The regulatory section (Section 5) is reasonably comprehensive but contains verbatim paragraph repetition (lines 451–453) are almost identically repeated at lines 454–457).
The conclusion mentions "cattle health" abruptly in a paragraph that opens with "The impact on cattle health is severely detrimental" — a confusing and likely erroneous statement.
Species-specificity of SFN delivery is addressed only superficially. Despite the title claiming relevance to animal feed, no systematic comparison of encapsulation performance across poultry, swine, ruminants, and aquaculture is provided with quantitative data.
Reference [79] cited in the context of SFN and pig hepatoprotection actually appears to be a study on selenium-enriched yeast in weaned pigs — a possible citation error.
Reference [80] cited for SFN dosage-efficacy relationships appears to be a porcine model of sepsis and glucocorticoid receptor function — also likely a citation mismatch.
The reference for the 2014 degradation kinetics study (ref. [85]) appears to be with incomplete bibliographic information, making independent verification difficult.
A thorough English language editing by a native speaker or professional service is recommended before this manuscript can be considered further.
The particle size stated in Table 1 for microfluidics-assisted self-assembly (5.3 nm) is extraordinarily small for chitosan-alginate nanoparticles and should be critically discussed, not presented uncritically.

Comments on the Quality of English Language

A thorough English language editing by a native speaker or professional service is recommended before this manuscript can be considered further.

Author Response

Comment 1: The review conflates general SFN nanoencapsulation literature (mostly from human pharmaceutical and food science contexts) with the stated focus on animal feed, which is actually covered by very few of the cited studies.

Response 1: We thank the reviewer for this comment. Indeed, most sulforaphane (SFN) nanoencapsulation literature comes from human pharmaceutical and food contexts, while studies in animal feed are very limited. This scarcity defines the research gap our work addresses. As detailed in Section 4.3 (“Species-Specific Delivery Challenges and Outcomes”), our focus is on encapsulating SFN to bypass rumen degradation. Additionally, SFN is light-sensitive and unstable under high pH, which can lead to degradation during feeding which will also negatively impact the bioavailability for non-ruminants, as supported by Figure 8 from the cited study. These challenges justify the need for feed-specific encapsulation strategies for livestock production.

Comment 2: The authors explicitly acknowledge on page 8 (lines 255–257): "there is a lack of precise studies that capitalize on encapsulating SFN using IG for animal feed" — which fundamentally undermines the review's premise and scope.

Response 2: We appreciate the reviewer’s observation regarding the limited number of studies on encapsulating sulforaphane (SFN) using ionic gelation (IG) for animal feed. We would like to clarify that this gap is precisely the novelty and motivation of our review. Our manuscript aims to synthesize existing knowledge from SFN encapsulation literature (mostly human and food studies) and highlight potential strategies for applying these methods to animal feed, thereby identifying opportunities for future experimental work in our laboratory. The review is intentionally proposed in nature, providing a conceptual framework for the design of SFN delivery systems for animals. This approach emphasizes the potential of IG and related encapsulation methods to enhance SFN stability and bioavailability in animal feed, which remains largely unexplored in the literature.

Comment 3: The review adds limited conceptual novelty beyond what existing SFN microencapsulation reviews already cover.

Response 3: We are grateful for the reviewer’s suggestion for this important comment. We agree that prior reviews have discussed sulforaphane microencapsulation, particularly in food and pharmaceutical contexts. However, the conceptual novelty of the present review lies in repositioning SFN encapsulation within an animal-feed framework, with emphasis on feed-stage instability, ruminal degradation, gastrointestinal pH sensitivity, and species-specific delivery challenges. These aspects are not the central focus of existing SFN microencapsulation reviews. To clarify this distinction, we have revised the manuscript to more explicitly define the review’s scope and novelty in the Introduction and Discussion.

Comments 4: Heading numbering is erratic and inconsistent throughout. Section 1 ("Introduction") is followed by another "1." (Ionic gelation as an engineering framework) then another “1” (Engineering stability: encapsulation design and protective performance). Then later sections appear as "1." again (Measurable efficacy in animal models, p. 10). This indicates the manuscript was assembled from separate components without proper integration.

Response 4: We acknowledge the reviewer’s concern regarding repeated section numbering. The numbering in our original Word document was sequential and correctly formatted. However, during conversion to PDF, the automatic heading numbering may have been misinterpreted by the PDF generator, leading to the perceived repetition. We have carefully reviewed the formatting, and in the revised manuscript, we have ensured that all headings and subheadings are consistently numbered and visually clear in both Word and PDF formats.

Comment 5: Sub-section numbering is mismatched with parent sections. For example, sub-sections labeled 2.2 and 2.3 appear before section 2 is introduced; section 3.1 appears before 3 is formally opened.

Response 5: We thank you for the concern about the mismatch with sub-sections and parent sections, once more, it seems when the word document was being converted to PDF the automatic heading numbering may have been misinterpreted by the PDF generator, leading to sub-section numbering mismatched with parent sections.

Comment 6: The abstract mentions chitosan and alginate as the primary focus, but the body expands broadly into liposomes, silk fibroin nanoparticles, PLGA, PEGylated nanoparticles, and dendrimers — far beyond ionic gelation.

Response 6: We appreciate the reviewer’s insightful comment. We have revised the manuscript to clarify that silk fibroin nanoparticles, PLGA, PEGylated nanoparticles, and nanoliposomes are discussed specifically for pre-stabilizing sulforaphane due to its instability. These systems serve as pre-stabilization steps before the synthesis of ionic-gelation-based carriers, as illustrated in Figure 4, maintaining ionic gelation as the primary encapsulation focus for feed-specific delivery.

Comment 7: Table 1 is titled "Gefitinib nanocarrier comparison" — Gefitinib is an anticancer drug with no relevance to sulforaphane or animal feed.

Response 7: We appreciate the reviewer’s observation. To better support the focus of our review, we have included a new Table 1 summarizing representative sulforaphane (SFN) encapsulation strategies, including microfluidics, micelle formation, emulsification, pre-gelation, and anti-solvent/composite nanoparticle approaches. This table highlights both direct SFN studies and related isothiocyanate systems that conceptually inform potential animal-feed applications. It clearly supports our focus on feed-specific delivery challenges, rumen bypass, and SFN stability, reinforcing the novelty and relevance of our review.

Comment 8: Several paragraphs (particularly in Section 3.4, pp. 9–10) discuss CpG oligonucleotides, immunotherapy, and cancer vaccine applications — topics entirely unrelated to the manuscript's stated scope.

Response 8: We appreciate the constructive feedback, upon careful revision, we agree that the discussion of CpG oligonucleotides, immunotherapy, and cancer vaccine applications extended beyond the central scope of this review. Our intention in citing Figure 5B was not to broaden the manuscript into immunotherapeutic applications, but to illustrate the broader carrier versatility of chitosan-based nanoparticles, particularly their capacity for bioactive loading, structural functionalization, and controlled-release delivery. To maintain stronger alignment with the manuscript’s focus, we have substantially reduced this discussion, removed most references to CpG ODNs, immunotherapy, and cancer vaccines, and revised the section to emphasize only the scope-relevant implication: that chitosan nanoparticles are adaptable, biocompatible carrier systems with potential value for stabilizing and delivering sulforaphane in feed-oriented formulations.

Comment 9: The regulatory section (Section 5) is reasonably comprehensive but contains verbatim paragraph repetition (lines 451–453) are almost identically repeated at lines 454–457).

Response 9: The authors are grateful for the insight on the repetitive text in Section 5 (lines 451–457). The redundancy has been removed and the content revised to eliminate duplication while maintaining the completeness of the regulatory discussion.

Comment 10: The conclusion mentions "cattle health" abruptly in a paragraph that opens with "The impact on cattle health is severely detrimental" — a confusing and likely erroneous statement.

Response 10: Upon careful revision, we recognize that the statement in the conclusion regarding “cattle health” was an oversight and potentially confusing. This sentence has been removed to maintain clarity and ensure that the conclusion accurately reflects the focus of the review.

Comment 11: Species-specificity of SFN delivery is addressed only superficially. Despite the title claiming relevance to animal feed, no systematic comparison of encapsulation performance across poultry, swine, ruminants, and aquaculture is provided with quantitative data.

Response 11: We really appreciate your concern with the limited livestock studies. Section 4.3 has been retained and slightly expanded to emphasize how SFN metabolism, stability, and bioavailability vary between ruminants and non-ruminants, and to include examples of observed outcomes in pigs and broilers. Additionally, we have included references that support the conditions under which sulforaphane undergoes degradation, highlighting factors such as pH and temperature that affect its stability. While additional livestock studies are currently limited, this highlights a clear gap in knowledge, which our research aims to address by systematically evaluating SFN delivery and stability in animal feed. We have acknowledged these constraints in the manuscript and emphasized the need for further research to validate SFN delivery and efficacy across diverse animal species.

Comment 12: Reference [79] cited in the context of SFN and pig hepatoprotection actually appears to be a study on selenium-enriched yeast in weaned pigs — a possible citation error.

Response 12: We recognize the reviewer’s point and have addressed it. This was a citation error, and we have corrected it. The previously cited reference on selenium-enriched yeast in weaned pigs has been removed from this context and replaced with the appropriate study by Wang et al., which evaluated sulforaphane in a neonatal piglet model of hypoxic–ischemic brain injury. We have also revised the text to clarify that the evidence relates to SFN-mediated neuroprotection and oxidative-stress modulation, rather than pig hepatoprotection. In that study, SFN increased nuclear Nrf2 and γ-glutamylcysteine synthetase expression and improved neuronal viability after HI injury.

Comment 13: Reference [80] cited for SFN dosage-efficacy relationships appears to be a porcine model of sepsis and glucocorticoid receptor function — also likely a citation mismatch.

Response 13: The reviewer’s comment was acknowledged and rectified. The reference [80] was a follow up reference to support reference [79], however after reviewing we realized the wrong reference was cited from the main source. We however removed the complete sentence and reference.

Comment 14: The reference for the 2014 degradation kinetics study (ref. [85]) appears to be with incomplete bibliographic information, making independent verification difficult.

Response 14: We thank the reviewer’s comment, as the bibliographic for reference [85] indeed was incomplete. The bibliographic for the said reference is now completed.

Comment 15: A thorough English language editing by a native speaker or professional service is recommended before this manuscript can be considered further.

Response 15: We appreciate the reviewer’s comment regarding language clarity. The manuscript has now undergone thorough English language editing by a professional service to improve grammar, readability, and overall presentation. We believe these revisions have enhanced the manuscript’s clarity and precision.

Comment 16: The particle size stated in Table 1 for microfluidics-assisted self-assembly (5.3 nm) is extraordinarily small for chitosan-alginate nanoparticles and should be critically discussed, not presented uncritically.

Response 16: The authors have acknowledged error made in then Table 1, we however completely removed the table and implemented a new table that reinforced the novelty and relevance of our review.

Reviewer 2 Report

Comments and Suggestions for Authors

biology-4313825

Ionic Gelation for Nano-Delivery of Sulforaphane in Animal Feed: A Critical Review of Stability, Efficacy, and Translation Potential

GENERAL REMARKS

The manuscript addresses a relevant and emerging topic, namely the application of ionic gelation for the nano-delivery of sulforaphane in animal feed. The integration of nanotechnology, bioactive compounds, and livestock nutrition is timely and potentially valuable. In particular, the focus on improving sulforaphane stability and bioavailability represents a meaningful contribution to the field. In its current form, however, the manuscript presents several conceptual, structural, and methodological limitations that reduce its scientific rigor and overall impact. Although the work is presented as a review, it lacks a clear and transparent methodological framework. There is no description of the literature search strategy, inclusion criteria, or synthesis approach. As a result, the manuscript reads more as a narrative compilation than a structured review, raising concerns about completeness, potential selection bias, and reproducibility. The interpretation of the literature also tends to exceed the level of available evidence. Across multiple sections, associative findings, often derived from in vitro or biomedical studies, are described using deterministic or causal language, particularly when linked to animal performance, health outcomes, or systemic effects. This weakens the scientific robustness of the manuscript and calls for a more cautious and evidence-aligned tone. From a structural perspective, the manuscript appears unbalanced and occasionally difficult to follow. Fundamental concepts, such as ionic gelation principles, are presented in considerable detail, while their direct connection to sulforaphane delivery in feed systems is not always clearly articulated. At the same time, sections on extraction technologies, cellular mechanisms, and regulatory aspects are interwoven without a clear hierarchical organization, resulting in redundancy and abrupt transitions. The scope of the review also appears partially diluted by the inclusion of content that is only marginally related to the central topic. Some sections focus extensively on biomedical pathways or nanotechnology applications that are not directly contextualized within livestock feeding systems, whereas aspects of greater practical relevance, such as in vivo validation, feed matrix interactions, and applicability under production conditions, are comparatively underdeveloped. Language and presentation further limit the readability of the manuscript. While the content is generally understandable, the text contains grammatical inaccuracies, unclear phrasing, inconsistent terminology, and occasional typographical issues. The overall tone is sometimes overly assertive relative to the supporting evidence. Overall, the manuscript has potential but requires substantial revision. Greater methodological transparency, improved alignment between evidence and interpretation, and a clearer structural organization would significantly strengthen the contribution. A more focused discussion on livestock-relevant applications and limitations would further enhance its scientific value.

My specific comments are listed below. I hope they will help to improve the quality of the manuscript.

SPECIFIC COMMENTS

L 18–27 (Abstract). The abstract is informative but tends to overstate outcomes (e.g., “enhanced immune responses” and “reduced oxidative stress”). These statements should be moderated to reflect the type of evidence available (e.g., “associated with” or “reported in experimental models”).

L 52–60. The manuscript claims a “clear gap” in the literature without providing a sufficiently structured or referenced justification. This statement should be supported with more explicit comparison to existing reviews.

L 72–84. The objectives are ambitious and include claims of establishing a “systematic framework,” yet no methodological structure is provided. This creates a mismatch between stated aims and actual approach.

L 86–118. The section on ionic gelation principles is scientifically correct but overly detailed relative to its contribution to the review’s novelty. Consider condensing and focusing on aspects directly relevant to sulforaphane delivery.

L 124–140. Statements regarding sulforaphane stability improvement through ionic gelation are presented as generally established, but supporting evidence is not critically discussed. Variability across encapsulation methods should be acknowledged.

L 155–174. The section on ultrasonic extraction appears only partially relevant to the main focus of ionic gelation-based delivery. Its inclusion should be better justified or reduced.

L 225–245. The claim that the proposed encapsulation strategy represents a “novel contribution” is not sufficiently supported. It is unclear whether this is a conceptual synthesis or a validated approach. This should be clarified and toned down if necessary.

L 254–260. The statement that ionic gelation “could serve as a reliable technique” is acceptable, but should explicitly acknowledge the limited availability of in vivo validation in livestock systems.

Table 1 (L 260–263). The table compares nanocarrier systems but includes compounds (e.g., gefitinib) not directly related to sulforaphane or animal feed. This reduces coherence and should be revised.

L 266–291. This section introduces CpG ODN delivery systems, which are not directly relevant to sulforaphane delivery in feed. This content appears off-topic and should be removed or clearly justified.

L 300–310. Statements about improved bioavailability and systemic delivery are plausible but lack critical discussion of experimental limitations (e.g., species differences, dosage, delivery conditions).

L 311–320. The description of Nrf2/NF-κB pathways is accurate but overly detailed for a feed-oriented review. Consider summarizing and focusing on relevance to animal systems.

L 328–351. The manuscript uses deterministic language when describing biological effects (e.g., “confirms its potent antioxidant capacity”). These statements should be moderated and clearly linked to the type of experimental model (in vitro vs. in vivo).

L 372–380. The link between sulforaphane supplementation and systemic physiological outcomes is presented as direct, but supporting livestock-specific evidence remains limited. This should be clearly acknowledged.

L 401–410. The discussion of species-specific differences is relevant and well introduced. However, it could be expanded with more concrete examples from livestock studies.

L 414–418. Claims regarding improved growth and productivity are potentially overstated and should be supported with more robust or species-specific data.

L 423–460. The regulatory section is informative but partially generic and not fully integrated with the specific case of sulforaphane nano-delivery. Consider tightening the focus.

L 484–503. The toxicity discussion is relevant but remains general. More emphasis should be placed on feed-specific exposure pathways and realistic risk scenarios.

L 524–548. The future perspectives section is overly focused on biomedical applications rather than livestock systems. The alignment with the stated scope of the review should be improved.

L 568–585 (Conclusion). The conclusion contains overgeneralized claims (e.g., impact on animal productivity and sustainability) that are not fully supported by the reviewed evidence. These should be moderated

Comments on the Quality of English Language

The language requires substantial revision. Issues include:

grammatical errors
inconsistent terminology (e.g., SFN, nanoparticles, delivery systems)
overly long and complex sentences
occasional typographical issues

A thorough language editing is strongly recommended.

Author Response

General response: We thank the reviewer for their thoughtful and constructive comments. All of the points raised have been carefully considered, and the manuscript has been revised accordingly. We are grateful for the time and expertise invested in reviewing this work.

My specific comments are listed below. I hope they will help to improve the quality of the manuscript.

SPECIFIC COMMENTS

Comment 1: L 18–27 (Abstract). The abstract is informative but tends to overstate outcomes (e.g., “enhanced immune responses” and “reduced oxidative stress”). These statements should be moderated to reflect the type of evidence available (e.g., “associated with” or “reported in experimental models”).

Response 1: Thank you for this helpful comment. We agree that the previous wording in the abstract could overstate the strength of the available evidence. The manuscript body discusses the supporting evidence in detail, including reported improvements in SFN bioavailability, absorption and experimental evidence for modulation of oxidative-stress, immune and inflammatory pathways. To avoid redundancy in the abstract while accurately reflecting the nature of the evidence, we have moderated the abstract wording from “enhanced nutritional absorption, elevated immune responses, and reduced oxidative stress in animals” to “associated with improved bioavailability, absorption and reported, mainly in experimental cellular and animal models, to modulate immune-related and oxidative-stress pathways.” This revision preserves the purpose of the abstract as a concise overview while avoiding overstatement.

Comment 2: L 52–60. The manuscript claims a “clear gap” in the literature without providing a sufficiently structured or referenced justification. This statement should be supported with more explicit comparison to existing reviews.

Response 2: We thank the reviewer for pointing this out. We have revised the manuscript to explicitly compare prior reviews, highlighting that while SFN encapsulation has been explored in human and rodent models (Mangla et al., 2021; Cascajosa-Lira et al., 2024; Houghton, 2019), there is limited systematic discussion of nanoencapsulation design and application for livestock feed. Evidence from pigs, broilers, and ruminants demonstrates that nanoencapsulated SFN can improve growth, liver function, and resilience, but species-specific challenges such as ruminal degradation remain and other factors that affected the stability (light via feeding and pH in GI tract) that negatively impact the bioavailability of sulforaphane in non-ruminant. This revision clarifies the nature of the literature gap and provides appropriate justification for our review’s focus. We utilized this method also to prevent the order of the reference as there are proof of this reference in the text and bibliographic.

Comment 3: L 72–84. The objectives are ambitious and include claims of establishing a “systematic framework,” yet no methodological structure is provided. This creates a mismatch between stated aims and actual approach.

Response 3: The authors thank you for this valuable comment. We agree that the previous wording could imply that a fully validated or systematic methodological framework had been established. Since this manuscript is a conceptual critical review rather than an experimental study or formal systematic review, we have revised the wording to clarify the nature of the framework. Specifically, we now describe it as a structured conceptual framework that integrates evidence on SFN instability, ionic gelation design principles, encapsulation performance, controlled release, species-specific digestive challenges, and regulatory considerations. We also clarified that the framework is intended as a design roadmap for future experimental validation in livestock feed systems, rather than as an already validated protocol.

Comment 4: L 86–118. The section on ionic gelation principles is scientifically correct but overly detailed relative to its contribution to the review’s novelty. Consider condensing and focusing on aspects directly relevant to sulforaphane delivery.

Response 4: We appreciate the suggestion to focus the section on content directly relevant to sulforaphane delivery. The revised section has been condensed to highlight key principles of ionic gelation such as polymer cross-linking, particle size, stability, and controlled release that are directly applicable to SFN encapsulation in animal feed. Extraneous details on unrelated applications have been removed, while references supporting the scientific principles remain. This ensures that the section provides necessary conceptual background while emphasizing its relevance to the review’s novelty.

Comment 5: L 124–140. Statements regarding sulforaphane stability improvement through ionic gelation are presented as generally established, but supporting evidence is not critically discussed. Variability across encapsulation methods should be acknowledged.

Response 5: Acknowledgment is given for the above comment. We have added specific references to support the statement that ionic gelation enhances the stability of sulforaphane. The revised sentence now reads: “Ionic gelation enhances the stability of encapsulated compounds such as sulforaphane [Zambrano et al., 2019], clearly linking the claim to published evidence. This maintains the sentence as a standalone advantage of ionic gelation while providing proper scientific support.

Comment 6: L 155–174. The section on ultrasonic extraction appears only partially relevant to the main focus of ionic gelation-based delivery. Its inclusion should be better justified or reduced.

Response 6: We thank the reviewer for the comment. We have revised the section on ultrasonic-assisted extraction (UAE) to focus on aspects most relevant to downstream ionic gelation-based delivery. The updated paragraph emphasizes that UAE enhances sulforaphane yield and purity, providing high-quality material necessary for effective encapsulation. The mechanistic basis, comparative efficacy, and scalability of UAE are briefly noted, supporting its inclusion as a preparatory step for ionic gelation without detracting from the review’s main focus.

Comment 7: L 225–245. The claim that the proposed encapsulation strategy represents a “novel contribution” is not sufficiently supported. It is unclear whether this is a conceptual synthesis or a validated approach. This should be clarified and toned down if necessary.

Response 7: Thank you for this helpful comment. We agree that the previous wording could imply that the proposed encapsulation strategy had already been experimentally validated as a complete protocol. We have revised the text to clarify that the novelty lies in the conceptual integration of individually reported steps into a unified SFN-loaded alginate–chitosan ionic gelation workflow. We now explicitly state that this proposed strategy should be regarded as a design framework for future validation rather than as an experimentally validated approach.

Comment 8: L 254–260. The statement that ionic gelation “could serve as a reliable technique” is acceptable, but should explicitly acknowledge the limited availability of in vivo validation in livestock systems.

Response 8: Thank you for highlighting this point. The authors agreed that the current literature on ionic gelation for SFN encapsulation in livestock is limited. While the manuscript demonstrates IG’s potential to improve stability and bioaccessibility, we have revised the text to explicitly acknowledge that in vivo validation in livestock systems is still scarce. This ensures that our claims are appropriately cautious and evidence-based.

Comment 9: Table 1 (L 260–263). The table compares nanocarrier systems but includes compounds (e.g., gefitinib) not directly related to sulforaphane or animal feed. This reduces coherence and should be revised.

Response 9: We appreciate the reviewer’s observation. To better support the focus of our review, we have included a new Table 1 summarizing representative sulforaphane (SFN) encapsulation strategies, including microfluidics, micelle formation, emulsification, pre-gelation, and anti-solvent/composite nanoparticle approaches. This table highlights both direct SFN studies and related isothiocyanate systems that conceptually inform potential animal-feed applications. It clearly supports our focus on feed-specific delivery challenges, rumen bypass, and SFN stability, reinforcing the novelty and relevance of our review.

Comment 10: L 266–291. This section introduces CpG ODN delivery systems, which are not directly relevant to sulforaphane delivery in feed. This content appears off-topic and should be removed or clearly justified.

Response 10: We appreciate the constructive feedback, upon careful revision, we agree that the discussion of CpG oligonucleotides, immunotherapy, and cancer vaccine applications extended beyond the central scope of this review. Our intention in citing Figure 5B was not to broaden the manuscript into immunotherapeutic applications, but to illustrate the broader carrier versatility of chitosan-based nanoparticles, particularly their capacity for bioactive loading, structural functionalization, and controlled-release delivery. To maintain stronger alignment with the manuscript’s focus, we have substantially reduced this discussion, removed most references to CpG ODNs, immunotherapy, and cancer vaccines, and revised the section to emphasize only the scope-relevant implication: that chitosan nanoparticles are adaptable, biocompatible carrier systems with potential value for stabilizing and delivering sulforaphane in feed-oriented formulations.

Comment 11: L 300–310. Statements about improved bioavailability and systemic delivery are plausible but lack critical discussion of experimental limitations (e.g., species differences, dosage, delivery conditions).

Response 11: Thank you for your feedback regarding the discussion of bioavailability and systemic delivery. We acknowledge that experimental limitations, including species differences, dosage, and delivery conditions, are important considerations. In the revised manuscript, we have now provided a detailed, referenced conceptual dosage framework for sulforaphane in livestock feed. The paragraph integrates oral administration evidence in rodents and humans, safety thresholds (LD50), analogous feed studies with 3‑NOP in cattle, and encapsulation technology with reported loading efficiencies. This presentation aims to give readers a practical, evidence-based guideline while clearly noting that in vivo studies are required to validate efficacy, safety, and species-specific responses. References are provided for all key points to support the plausibility of bioavailability and systemic delivery.

Comment 12: L 311–320. The description of Nrf2/NF-κB pathways is accurate but overly detailed for a feed-oriented review. Consider summarizing and focusing on relevance to animal systems.

Response 12: We thank the reviewer for the suggestion. We have carefully reviewed the manuscript and removed unnecessary details from the Nrf2/NF-κB pathway section, retaining only the relevant information supported by references and figures as needed. This revision ensures the section is concise and focused on its relevance to animal feed applications.

Comment 13: L 328–351. The manuscript uses deterministic language when describing biological effects (e.g., “confirms its potent antioxidant capacity”). These statements should be moderated and clearly linked to the type of experimental model (in vitro vs. in vivo).

Response 13: We have carefully revised the manuscript to moderate such statements, replacing terms like “confirms” with “suggests”, and explicitly linking each observed effect to the experimental model (in vitro vs. in vivo). This ensures that the reported effects accurately reflect the evidence while maintaining clarity and scientific rigor.

Comment 14: L 372–380. The link between sulforaphane supplementation and systemic physiological outcomes is presented as direct, but supporting livestock-specific evidence remains limited. This should be clearly acknowledged.

Response 14: We appreciate the reviewer’s suggestion regarding the evidence for systemic physiological outcomes in livestock. We have revised the manuscript to acknowledge that direct livestock-specific studies remain limited. Statements implying a direct effect of SFN on animal health have been moderated or removed, and we now explicitly indicate when results are derived from in vitro models or preliminary studies. This ensures that conclusions are appropriately linked to the experimental evidence, highlighting the need for further validation in diverse livestock species.

Comment 15: L 401–410. The discussion of species-specific differences is relevant and well introduced. However, it could be expanded with more concrete examples from livestock studies.

Response 15: We really appreciate your concern with the limited livestock studies. Section 4.3 has been retained and slightly expanded to emphasize how SFN metabolism, stability, and bioavailability vary between ruminants and non-ruminants, and to include examples of observed outcomes in pigs and broilers. Additionally, we have included references that support the conditions under which sulforaphane undergoes degradation, highlighting factors such as pH and temperature that affect its stability. While additional livestock studies are currently limited, this highlights a clear gap in knowledge, which our research aims to address by systematically evaluating SFN delivery and stability in animal feed. We have acknowledged these constraints in the manuscript and emphasized the need for further research to validate SFN delivery and efficacy across diverse animal species.

Comment 16: L 414–418. Claims regarding improved growth and productivity are potentially overstated and should be supported with more robust or species-specific data.

Response 16: We thank the reviewer for this important comment. We have revised the text to state that the findings come from "some studies" and now explicitly note that "these findings require further studies for confirmation and more species-specific data." The claim has also been softened from active reporting to "has been associated with." We believe these revisions adequately address the concern regarding overstated claims.

Comment 17: L 423–460. The regulatory section is informative but partially generic and not fully integrated with the specific case of sulforaphane nano-delivery. Consider tightening the focus.

Response 17: This comment is well taken. We have revised the regulatory section to explicitly integrate the case of nano-encapsulated sulforaphane throughout, including specific references to SFN in relation to FDA evaluation, EFSA pre-market approval, and existing data gaps. Duplicate text has also been removed. The section is now more tightly focused on SFN nano-delivery rather than generic nanotechnology regulation.

Comment 18: L 484–503. The toxicity discussion is relevant but remains general. More emphasis should be placed on feed-specific exposure pathways and realistic risk scenarios.

Response 18: The authors have acknowledged the reviewer for emphasizing the need to focus on feed-specific exposure pathways and realistic risk scenarios. In the revised manuscript, Section 3.5 presents a conceptual dosage framework for sulforaphane, including median effective doses in mice, human oral dose ranges, and references to LD₅₀ data. This ensures that potential toxicity is evaluated in the context of realistic oral exposure via feed. We also note conditions under which SFN may degrade and highlight the safety limits, emphasizing the practical relevance to animal feed applications.

Comment 19: L 524–548. The future perspectives section is overly focused on biomedical applications rather than livestock systems. The alignment with the stated scope of the review should be improved.

Response 19: We have revised the section to clarify that while some nanocarriers were originally developed for pharmaceutical applications, they are discussed conceptually to illustrate strategies for controlled release, enhanced bioavailability, and protection from degradation. We have emphasized that these principles could be adapted for livestock feed applications, while the section now primarily focuses on food-grade, feed-relevant delivery systems (e.g., chitosan-alginate hydrogels, gelatin-pectin microcapsules, basil seed gum coatings, and plant-derived vesicles).

Additionally, any reference to human models has been updated to highlight livestock-specific in vivo validation, ensuring the discussion aligns with animal health and practical feed applications. These changes clarify the relevance of the nanocarrier discussion to livestock feed while maintaining the conceptual insights from pharmaceutical research.

Comment 20: L 568–585 (Conclusion). The conclusion contains overgeneralized claims (e.g., impact on animal productivity and sustainability) that are not fully supported by the reviewed evidence. These should be moderated.

Response 20: We appreciate this observation. The conclusion has been carefully revised to avoid overgeneralization. Specifically, we have removed statements concerning cattle health, pollution mitigation, and reduced antibiotic reliance, as these were not adequately supported by the reviewed literature. Where references to productivity and sustainability remain, they have been qualified with cautious language (e.g., "suggested," "preliminary," "may contribute to," "pending further validation"). We believe the revised conclusion now presents a more balanced reflection of the current evidence.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thanks for the efforts exerted by the authors to improve the manuscripts and address all the comments

Author Response

Comment: Thanks for the efforts exerted by the authors to improve the manuscripts and address all the comments.

Response: We sincerely thank the reviewer for the positive evaluation and constructive guidance throughout the review process. The reviewer’s comments were very helpful in improving the clarity, balance, and scientific rigor of the manuscript. We are grateful that the revisions have strengthened the manuscript and helped bring it closer to a publishable standard.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The revised manuscript has improved substantially compared with the previous version, addressing several of the major concerns raised during the first round of review. Indeed, the revised version presents a more balanced narrative, moderates some of the original claims, better acknowledges the limited availability of direct in vivo evidence, and more clearly positions the proposed ionic gelation approach as a conceptual roadmap rather than as an experimentally validated protocol. These revisions strengthen the overall coherence and scientific transparency of the work.

Nevertheless, in my opinion, some aspects would still benefit from further refinement before publication, according to the points summarized below.

Although the conceptual positioning of the manuscript is now clearer, the distinction between experimentally demonstrated outcomes, mechanistic interpretations, and future translational opportunities should remain consistently explicit throughout the text. In several passages, conclusions still appear to extend beyond the strength of the currently available evidence, particularly where observations originating from cellular systems, murine studies, or human literature are translated into expected responses under livestock conditions. A final revision of the wording to maintain closer alignment between the level of evidence and the resulting interpretation would further strengthen the manuscript.

The section proposing a conceptual dosage framework remains the area where the manuscript appears least robust. The suggested livestock inclusion range seems to derive from extrapolation across heterogeneous biological systems rather than from direct dose–response evidence obtained in target animal species. While this approach may still be acceptable within the conceptual nature of the review, the section would benefit from being more explicitly presented as an illustrative example of formulation logic rather than as a recommendation applicable under field conditions.

The expanded discussion on livestock applications and species-specific considerations is appreciated and increases the translational relevance of the review. Nevertheless, several arguments supporting expected efficacy continue to rely predominantly on indirect evidence. The discussion would therefore benefit from maintaining a clearer separation between observed biological mechanisms and expected productive outcomes, avoiding the impression that improvements in encapsulation efficiency necessarily translate into measurable field performance.

Author Response

Nevertheless, in my opinion, some aspects would still benefit from further refinement before publication, according to the points summarized below.

Comment 1: Although the conceptual positioning of the manuscript is now clearer, the distinction between experimentally demonstrated outcomes, mechanistic interpretations, and future translational opportunities should remain consistently explicit throughout the text. In several passages, conclusions still appear to extend beyond the strength of the currently available evidence, particularly where observations originating from cellular systems, murine studies, or human literature are translated into expected responses under livestock conditions. A final revision of the wording to maintain closer alignment between the level of evidence and the resulting interpretation would further strengthen the manuscript.

Response 1: The authors are grateful to the reviewer for this observation. We agree that clear separation between experimentally demonstrated findings, mechanistic interpretations, and translational hypotheses is essential. Accordingly, we have revised several sections throughout the manuscript to consistently distinguish between evidence derived from in vitro systems, rodent studies, and livestock investigations. Statements implying direct livestock outcomes have been moderated and rephrased to emphasize that such outcomes remain hypothetical until validated in target animal species.

Comment 2: The section proposing a conceptual dosage framework remains the area where the manuscript appears least robust. The suggested livestock inclusion range seems to derive from extrapolation across heterogeneous biological systems rather than from direct dose–response evidence obtained in target animal species. While this approach may still be acceptable within the conceptual nature of the review, the section would benefit from being more explicitly presented as an illustrative example of formulation logic rather than as a recommendation applicable under field conditions.
Response 2: We appreciate the reviewer's concern regarding the conceptual dosage framework. The intention of this section was not to provide practical feeding recommendations but rather to illustrate how information from different biological systems may be integrated into a preliminary formulation rationale. To avoid overinterpretation, we have revised the section to clearly state that the proposed range is a hypothetical example intended to demonstrate formulation logic and should not be interpreted as a validated livestock dosage recommendation.

Comment 3: The expanded discussion on livestock applications and species-specific considerations is appreciated and increases the translational relevance of the review. Nevertheless, several arguments supporting expected efficacy continue to rely predominantly on indirect evidence. The discussion would therefore benefit from maintaining a clearer separation between observed biological mechanisms and expected productive outcomes, avoiding the impression that improvements in encapsulation efficiency necessarily translate into measurable field performance. Changes are made between L316-318.

Response 3: We thank the reviewer for this valuable comment. To address this concern, we clarified the distinction between formulation-level evidence and expected livestock performance outcomes. Specifically, we revised the discussion to state that the proposed encapsulation strategy should be interpreted as formulation-level evidence supporting SFN delivery systems rather than direct proof of improved livestock performance. We also clarified that most current data are derived from in vitro systems, pharmaceutical models, or limited animal studies, and therefore do not yet confirm predictable outcomes under practical feeding conditions. These changes were made at L316–323 and L356–359