Preparation of Targeted Delivery Materials and Their Application in Animal Production

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review summarizes the main types, preparation techniques, and evaluation methods for targeted delivery materials, as well as their applications in animal production. It aims to provide theoretical references for the further technological implementation of these materials. 

The review is not combrehansive and other methods used were not included. Tables should be added to summarize studies. More illustrations are needed. A major revsion for the review is suggested.

Abstract: This is too general; revise to include more information about the methods.

Introduction: reorganize to have a well defined paragraphs. The ideas within the same paragraph is mixed up. There is lack of proper citations in the introduction and throughout the paper.

The methods used are not comprehansive, there are other methods that were ignored.

Provide illistrations for the method used.

The review has incomplete discussion of ideas, you need to revise to provide a comprehansive discussion, and provide your own assessements. For example, L161-163: Different microcapsule preparation methods each have their own advantages and disadvantages, and are suitable for encapsulation requirements of various types of active substances, thereby offering additional optional strategies for the targeted delivery of nutrients. What are the advantages and disadvanteges? What are the additional options? This applies to diffrent parts of the review.

3.2 and 3.3: check the titles

4. The Application of Targeted Delivery Technology in Animal Production. This section needs more information and more studies should be included. You need to extend your search to include more studies, you can create tables to summaries the data. Provide experiments that used some of these products. For example: Effects of Nano-Emulsified Vegetable Oil and Betaine on Growth Traits and Meat Characteristics of Broiler Chickens Reared under Cyclic Heat Stress. Animals 11(7):1911. DOI: 10.3390/ani11071911. 

 

 

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant and emerging topic: the use of targeted delivery carriers such as nanoparticles, microcapsules and gels/hydrogels to protect nutrients, probiotics, organic acids, essential oils, vaccines and drugs, and to improve their release at specific gastrointestinal or tissue sites in animal production. The review is potentially useful because it covers carrier types, preparation methods, evaluation approaches and applications in poultry, pigs and ruminants. However, the manuscript requires major revision before publication.

Major issues

1. The review lacks a literature-search method. The manuscript is presented as a review, but it does not describe how the literature was selected. There is no information on databases searched, search terms, time period, inclusion/exclusion criteria, animal species considered, or whether the review is narrative, scoping or systematic.
2. The scope is too broad and not sufficiently critical. The title refers broadly to targeted delivery materials and animal production. The manuscript covers nanoparticles, microcapsules, gels, preparation technologies, evaluation methods, poultry, pigs and ruminants. This breadth is useful, but the review often lists examples without critically comparing their relevance, maturity, cost, safety or scalability.
3. Too many examples are extrapolated from non-livestock models. Several sections rely on mouse or rat work to support evaluation methods or targeted delivery claims. These examples may be useful for explaining methodology, but they should not be presented as direct evidence for animal production. The authors should explicitly state when evidence comes from laboratory-animal or biomedical models.
4. Animal-production evidence needs deeper synthesis. The poultry, pig and ruminant sections provide useful examples; however, these are presented mainly as isolated examples. The authors should compare outcomes across species and target compounds, including dose, route of administration, target site, carrier type, release mechanism, performance response, immune response, microbiota effect and limitations.
5. Practical feed-manufacturing feasibility is underdeveloped. The manuscript states that targeted delivery materials can protect nutrients during feed processing and digestion. However, it does not sufficiently discuss whether these systems tolerate pelleting, extrusion, mixing, storage, humidity, heat, abrasion, transport and long-term shelf life. For animal production, these practical issues are central. A carrier that works in a laboratory gavage model may not survive commercial feed manufacture.
6. Cost and scalability are not adequately addressed. The conclusion mentions the need for low-cost, stable and scalable carrier materials. This is important, but it appears only at the end. The manuscript should critically assess cost and scalability throughout the review.
7. Safety, residue and regulatory issues are insufficiently discussed. The manuscript briefly mentions biocompatibility, low toxicity and biodegradability as desirable carrier properties. However, the review needs a dedicated safety section. Targeted delivery materials may affect gut microbiota, mucosal immunity, absorption kinetics, tissue residues, environmental excretion, milk/meat/egg safety, antimicrobial resistance, nanoparticle accumulation and consumer acceptance.
8. The review needs summary tables such as (examples): Carrier types and preparation methods: nanoparticles, microcapsules, gels/hydrogels; preparation method; suitable active compounds; advantages; disadvantages; livestock relevance; Evaluation methods: particle size/morphology, encapsulation efficiency, release kinetics, simulated digestion, rumen simulation, fluorescence imaging, in vivo performance trials; strengths and limitations; Animal-production applications: species, active compound, carrier, target site, dose, route, main outcome, evidence level.
9. Conclusions overstate application potential. The conclusion should emphasise that many systems remain experimental, that livestock-specific validation is limited, and that long-term safety, cost-effectiveness and feed-processing compatibility need further study.

Minor issues

Lines 37–43: The opening paragraph is relevant, but “amino and fatty acids” should be “amino acids and fatty acids”.

Lines 44–48: The statement that ruminal microorganisms degrade proteins, amino acids and polyphenols is correct, but the phrase “preventing the effective components from reaching the hindgut smoothly” is awkward. Use “preventing active compounds from reaching the intended post-ruminal site”.

Lines 48–52: The colitis example is biomedical rather than animal-production focused. Clarify why it is relevant to livestock.

Lines 53–58: TDDS are introduced from pharmaceutical science. This is useful, but the review should explain how livestock feed delivery differs from human drug delivery.

Lines 61–66: The sentence on polyunsaturated fatty acids is confusing. The problem is ruminal biohydrogenation of unsaturated fatty acids, not “poor hydrogenation”.

Lines 63–66: The example of chitosan-encapsulated flaxseed oil in dairy cows is highly relevant. Expand its practical significance and limitations.

Lines 78–88: Good general description, but “targeted recognition of specific intestinal sites or cells” may overstate many passive systems. Some nanoparticles protect or delay release without active recognition.

Lines 90–95: The lutein nanoparticle preparation is detailed. Consider shortening technical steps and emphasising why it matters for animal nutrition.

Lines 99–107: Emulsion solvent evaporation for butyrate and propionate is relevant, but discuss solvent residues and feed-scale feasibility.

Lines 108–112: Spray drying is practical for feed applications; discuss thermal stability of sensitive actives.

Figure 1: The figure is clear and visually useful, but some labels are small. Increase font size and standardise wording.

Lines 121–124: The statement that microcapsules have a liquid core may be too narrow. Some microcapsules contain solid or semi-solid cores.

Lines 125–129: pH-responsive release is important. Add examples of pH differences among stomach, rumen, abomasum, small intestine and colon.

Lines 151–160: Complex coacervation for walnut oil is relevant, but this is a food-material example. Clarify livestock application potential.

Lines 173–178: Enrofloxacin-loaded nanogels involve a veterinary drug. Discuss residue and withdrawal implications.

Lines 180–190: Gas shear-assisted ionic crosslinking is interesting but may not be scalable for feed. Add cost/equipment limitations.

Lines 190–197: Water-in-oil emulsion with calcium crosslinking is relevant. Add potential concerns about residual oil phase or surfactant.

Lines 214–245: Particle size, PDI and morphology are important, but the examples are mainly mouse/rat or biomedical. Clarify relevance to livestock.

Line 222: “SNX10-shRNA plasmid nanoparticles” may be outside the scope of animal production. Use as a methodological example only.

Lines 230–242: The nanomineral microcapsule example is useful, but it was administered to rats. Avoid implying direct livestock efficacy.

Lines 247–255: Good explanation of simulated gastric fluid. For ruminants, simulated rumen and abomasum conditions should be discussed in more detail.

Lines 256–267: Methane-reduction nanoparticle example is relevant to ruminants, but it is based on rumen-fluid simulation. Do not overstate in vivo applicability.

Lines 268–278: Lactic acid bacteria microcapsule example is one of the stronger ruminant-relevant examples. It should be highlighted.

Lines 298–335: This section is useful methodologically but mostly based on mouse gavage models. State clearly that these are preclinical evaluation methods and not direct livestock-production evidence.

Figure 2: The figure is visually useful but very detailed. Increase text size and ensure all labels are readable.

Lines 393–396: Avoid saying targeted delivery can “effectively address” intestinal parasites and antimicrobial resistance. Use more cautious wording.

Lines 436–440: Future research in piglets is appropriate, but also mention weaning diarrhoea, microbiota stability and feed intake.

Lines 447–452: Probiotic microcapsules in dairy cows are relevant, but evidence that they were released specifically in the distal intestine should be supported with direct recovery or marker data.

Lines 462–472: The Toxoplasma gondii nanoparticle vaccine in sheep is relevant for animal health but not feed delivery. Place it under veterinary applications rather than nutrition.

Lines 485–495: The conclusion is clear but broad. Add that evidence is uneven among species and applications.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Final Decision Recommendation: Major Revision

 

This review addresses delivery carriers for nutrients and bioactive compounds in animal production, and the manuscript has a generally clear structure. However, major revisions are required because of unclear terminology, inconsistent carrier classification and research scope, incomplete evaluation criteria, insufficient critical synthesis, limited coverage of ruminant nutrition, and multiple citation errors.

 

1. The manuscript uses the term “targeted delivery” to encompass protective encapsulation, sustained or controlled release, stimuli-responsive release, preferential accumulation at specific gastrointestinal sites, and active cellular targeting, without adequately distinguishing these concepts. These delivery modes should be clearly defined and discussed according to the level of evidence required to support each claim. In several cases, changes in intestinal microbiota or animal growth performance are interpreted as evidence of successful targeted delivery. However, such biological outcomes alone do not demonstrate site-specific delivery. Therefore, definitive expressions such as “confirmed”, “demonstrated”, and “successfully targeted” should be avoided unless supported by direct evidence, such as tracer-based localization, recovery or quantification of the cargo at the claimed target site, or validated receptor-mediated cellular uptake.

 

2. The Abstract and Introduction focus on oral gastrointestinal delivery, whereas Section 4 includes injected vaccines, intranasal vaccines, and intramammary treatments. The authors should either restrict the review to gastrointestinal delivery or revise the title, objectives, and structure to cover multiple administration routes.

 

3. The manuscript does not explain how studies were searched and selected. The authors should provide the databases, search terms, publication period, and inclusion and exclusion criteria, and clarify whether this is a narrative or systematic review.

 

4. Nanoparticles, microcapsules, and gels are not mutually exclusive categories. The authors should distinguish size, structure, material type, and release mechanism, and clarify the differences among nanogels, microgels, microcapsules, microspheres, and matrix microparticles.

 

5. Common feed-delivery systems, including liposomes, nanoemulsions, cyclodextrin complexes, lipid carriers, and conventional rumen-protected coatings, are not systematically discussed. These systems should be included or their exclusion should be justified.

 

6. Nanoprecipitation, extrusion, and electrospray are mentioned in the summary of Section 2 but are not adequately described in the main text. Each listed method should be introduced and compared, or removed from the summary.

 

7. Section 3 mainly discusses particle size, simulated digestion, and fluorescence imaging. It should also cover encapsulation efficiency, loading capacity, release kinetics, processing and storage stability, bioavailability, safety, and industrial scalability.

 

8. Section 3.3 is titled “In vitro simulation,” but its content concerns in vivo fluorescence imaging. The title should be changed to “In vivo Fluorescence Imaging.”

 

9.The manuscript mainly summarizes individual studies one by one. More direct comparisons are needed regarding carrier suitability, target sites, cargo types, advantages, limitations, safety, cost, and practical application.

 

10. The manuscript does not clearly explain how it differs from existing reviews on encapsulation and nanodelivery in animal production. The authors should state the unique framework and contribution of this review.

 

11. The ruminant section does not adequately address the rumen-degradation problem emphasized in the Introduction. The authors should add mainstream applications such as rumen-protected amino acids, choline, fatty acids, plant extracts, probiotics, enzymes, and methane-mitigation carriers.

 

12. The manuscript lacks a table comparing carrier types, preparation methods, release mechanisms, target sites, advantages, and limitations. An original conceptual figure distinguishing protection, controlled release, passive targeting, and active targeting should also be added.

 

13. Several in-text author names do not match the corresponding references, including Refs. 15, 16, 17, 22, 35, 37, and 39. The authors should check all in-text citations, reference numbers, author names, and attributed conclusions.

 

14. All non-standard abbreviations, gene symbols, and protein names should be defined at first use and used consistently throughout the manuscript.

 

15. The captions of Figures 1 and 2 should explain the main mechanisms and identify the source of each illustrated procedure. Figure 2 should clarify that its evaluation steps are derived from different studies rather than one continuous experiment.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

This review covers the preparation, evaluation, and application of targeted delivery materials (nanoparticles, microcapsules, gels) in poultry, pig, and ruminant production. The topic is timely and relevant to the journal's scope, the structure is logical, and the review is enriched by detailed, worked examples of preparation protocols rather than superficial citation lists. However, the manuscript contains a duplicated section heading, a recurring and systematic error in in-text author citation (given name used instead of surname in multiple instances), and an imbalance in the ruminant application section relative to the paper's own stated rationale. I recommend minor revision to address these issues along with several content gaps noted below.

1- The manuscript contains two sections both numbered/titled "3.2. In vitro simulation" and "3.3. In vitro simulation" (page 8), though the second one actually discusses in vivo fluorescence imaging. Please correct the heading (likely 3.3. In vivo fluorescence imaging or evaluation) and verify all subsequent cross-references and numbering are consistent throughout the manuscript.

2-Multiple in-text citations appear to use a given name rather than the surname, creating a mismatch with the reference list. Examples identified:

• "Veeresh et al. [15]" in text vs. "Toragall, V." in reference 15
• "Suzana et al. [16]" in text vs. "Carvalho, S.G." in reference 16
• "Lena et al. [17]" in text vs. "Spindler, L.M." in reference 17
• "Eric et al. [37]" in text vs. "Altermann, E." in reference 37
• "Yi et al. [39]" in text vs. "Li, Y." in reference 39

Please systematically audit all in-text citations against the reference list to identify and correct any additional instances of this error, as it will impede readers' ability to locate cited sources.

3- The Introduction motivates the review by emphasizing that ruminal microbial degradation prevents nutrients from reaching the hindgut intact, and cites Besharati et al. (ref 7, flaxseed oil biohydrogenation) as a key example of this problem. However, Section 4.3 does not revisit rumen-protected nutrient delivery (e.g., protected fats, amino acids, or proteins) at all; instead it presents examples of mastitis treatment (mammary gland target) and a nasal vaccine (mucosal immune target), which are not examples of overcoming ruminal degradation. Please add at least one concrete example of rumen-bypass nutrient encapsulation to align this section with the paper's stated motivation, or otherwise clarify/reframe the scope of Section 4.3.

4- Absence of cost, scalability, and regulatory considerations
Given that the Simple Summary explicitly targets feed manufacturers and livestock producers as an audience, the manuscript would benefit from at least a brief discussion of production cost, scalability of laboratory-scale preparation methods (e.g., spray-drying vs. small-batch ionotropic gelation) to industrial volumes, and the regulatory approval status of these materials as feed additives in major markets. This is currently absent throughout the manuscript.

5- The manuscript lack of negative/contradictory findings
Nearly all cited primary studies report successful, positive outcomes. For a balanced review, particularly one whose Conclusion raises concerns about long-term safety and ecological impact of these materials, please include at least a few examples of encapsulation failures, unexpected toxicity, or null results from the literature to avoid the impression of selective/positive-outcome reporting bias.

6- The manuscript has an internal inconsistency regarding cost claims
Line 57 states that targeted drug delivery systems (TDDS) "reduce overall costs" compared with conventional delivery, but Section 5 (Conclusions) states that developing "low cost" specialized carrier materials remains a future goal, implying current costs are not yet low. Please reconcile this apparent contradiction or clarify that the cost reduction refers specifically to reduced dosing/drug loss rather than overall production cost.

7- Reference 39 — author listed as "Liu, I." appears to be a probable typographical error (unusual initial); please verify against the original source (likely "Liu, L." or similar).

8- Section 2.1, nanoparticle size definition, the 1–100 nm range is stated as a strict definition, but several cited "nanoparticle" systems in the review may exceed this range in practice; consider a brief acknowledgment of this common definitional fuzziness in the field.

9- Future-perspective statements are repeated across multiple subsections (end of 3.2, end of 4.2, end of 4.3, and Section 5) with similar generic content (e.g., "future research should..."). Consider consolidating these into a single, more specific roadmap, ideally distinguishing near-term vs. long-term priorities to reduce redundancy and increase actionable value for readers.

10- Table format for method comparison: Section 2's closing paragraph (lines 197–212) summarizes preparation method suitability in dense prose (which method suits which active substance type). Consider converting this into a summary table for easier reference by readers (e.g., feed manufacturers scanning for the appropriate method for their specific compound).

11- Figure 2 caption 

Please confirm the figure caption ("Characterization, in vitro release, and in vivo fluorescence evaluation of probiotic-encapsulated hydrogel beads") accurately reflects all panels shown, as the in-text description (lines 344–348) references three complementary methods that should each be clearly identifiable in the figure.

12- Line 63–66 (Besharati et al. example), the percentage supplementation (7% chitosan-encapsulated flaxseed oil) is stated, but the actual magnitude of biohydrogenation reduction achieved is not quantified in the text; adding the effect size would strengthen this as a lead example.

13- Section 4.1, line 370, "27-fold increase" in fluorescence signal is a striking figure; please confirm this value is correctly transcribed from the source study and consider briefly noting the comparison baseline for clarity (unlabeled nanoparticles vs. free dye).

Comments on the Quality of English Language

 The English could be improved to more clearly express the research.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 5 Report

Comments and Suggestions for Authors

The manuscript provides a comprehensive and well-organized overview of targeted delivery materials and their application in animal production. The topic is timely and highly relevant, particularly considering the growing interest in precision nutrition, reduction of antimicrobial use, and sustainable livestock production. The review is clearly structured, the figures are informative, and the literature cited is generally recent and appropriate.

From the perspective of veterinary science, histology, and animal welfare, I have several suggestions that could further strengthen the manuscript.

- Expand the discussion on tissue-level evaluation of targeted delivery systems.

Although the manuscript appropriately describes physicochemical characterization, in vitro simulation, and in vivo fluorescence imaging, the role of histopathology is only briefly mentioned. Histological examination is one of the most important approaches for confirming the biological safety and effectiveness of targeted delivery systems. Besides demonstrating localization of the carrier, histology enables assessment of epithelial integrity, inflammatory responses, immune-cell infiltration, goblet cell distribution, mucosal architecture, and possible tissue toxicity. Including a short paragraph emphasizing the importance of histological evaluation would provide a more balanced overview of the available assessment methods.

- Include greater consideration of animal welfare (3Rs).

Targeted delivery technologies have the potential not only to improve production efficiency but also to enhance animal welfare by reducing disease incidence, lowering therapeutic doses, decreasing repeated treatments, and minimizing handling-associated stress. Furthermore, advanced in vitro gastrointestinal models discussed in the manuscript may contribute to the Replacement and Reduction principles of the 3Rs by decreasing the number of experimental animals required during material optimization. A brief discussion of these aspects would broaden the impact of the review and better align it with the scope of Animals.

- Clarify limitations of current in vivo studies.

Many cited studies demonstrate promising biological effects; however, most are relatively short-term and focus on specific experimental models. It would be valuable to emphasize that long-term safety, chronic tissue responses, biodistribution, biodegradation, and possible accumulation of carrier materials remain insufficiently investigated, particularly under commercial livestock production conditions.

- Improve consistency of terminology.

Throughout the manuscript, the terms "targeted delivery materials", "targeted delivery carriers", "carriers", and "delivery systems" are used somewhat interchangeably. Selecting one preferred term and using it consistently would improve readability.

- Minor editorial issue.

Section numbering appears to contain an error. The second subsection titled "3.3. In vitro simulation" actually discusses in vivo fluorescence imaging and should therefore be renamed accordingly.

Overall, this is a valuable and well-written review that summarizes an emerging field with considerable importance for veterinary medicine and animal production. The suggested revisions are relatively minor and primarily intended to improve the translational perspective, particularly regarding histological evaluation and animal welfare considerations. After these revisions, the manuscript will provide an even stronger contribution to the literature.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for providing a revised review; you improved the review significantly. However, there are still some issues with citations, especially in the revised parts. You need to work on the distribution of the citations; sometimes you have more than one citation in other parts, and no citation at all. More citations on practical applications are needed. I provided 2 papers; you can use them or find more.

L57-60: Add references

L71-76: Add references

L86: Add references

L89-94: Add references

L117: Add references

L157-161: Add references

L167-174: Add references

L199-203: Add references

L231-235: Add references and elaborate

L295-301: Add references

L310-318: Add references

L327-328: Add references

L381-390: Add references

L393-398: Add references

L424-435: Add references

This applies to the remainder of the review as well.

 

 

 

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made substantial efforts to address the comments raised in the previous review, and the manuscript has improved considerably. Nevertheless, several important issues raised in the previous review remain incompletely resolved. In addition, the revision has introduced or retained a number of inconsistencies in terminology, tables, section numbering and figure cross-referencing. I therefore recommend major revision before the manuscript can be considered for publication.

Major issues

1. The literature-search methodology remains insufficiently reproducible. I appreciate the addition of Section 2 and the explicit clarification that this is a narrative rather than systematic review. However, the current Methods section still does not provide a sufficiently transparent account of how the evidence base was assembled. The authors state that Web of Science, ScienceDirect, PubMed, Scopus and the Google search engine were searched using keyword combinations that “included, but were not limited to” targeted delivery, carriers, in vitro simulation, microcapsules and animal production. They further state that the strategy was adjusted according to each search engine. This does not permit the search to be reproduced. The numerical description is also unclear. The manuscript states that approximately 268 records were initially retrieved, that 116 were selected after title and abstract screening, and that “another 101 records were chosen after a full-text evaluation”. Presumably, 101 of the 116 records were retained after full-text assessment, but the present wording could be interpreted as 101 additional records.
2. Several of my previous comments are not actually answered in the response letter. A point-by-point response should indicate what was changed, where it was changed, or why the authors decided not to make the requested change. Several substantive comments instead receive only “Thank you for your positive feedback”, although they were not simply positive comments. Please give substantive responses.
3. The distinction between passive protection/release and true active targeting remains conceptually unclear. My previous Comment 16 has not been adequately addressed. The revised manuscript still states that the delivery mechanism of nanoparticles “involves targeted recognition of specific intestinal sites or cells through surface modification”. This is applicable to some actively targeted systems, but not to nanoparticles in general. Many systems discussed in this manuscript primarily: protect the active compound from degradation; delay release; respond to pH, enzymes or microbial conditions; or increase residence time or mucosal exposure. These mechanisms should not automatically be described as active recognition or precise targeting. Please revise the general definition so that active receptor- or ligand-mediated targeting is clearly distinguished from passive localisation, environmentally triggered release, rumen protection and controlled release.
4. The animal-production summary tables are useful but still do not fully address the previous request. Tables 5–7 are a valuable addition and substantially improve the manuscript. The revised application tables include several elements but still omit dose and do not contain a consistent evidence-level/study-design field. Dose is particularly important. For example, the text reports 400 and 700 mg/kg microencapsulated hydrolysable tannins, but this information disappears from Table 5. I strongly recommend adding at least: Dose/concentration; study design/evidence type; duration (where relevant). The evidence-type column could use straightforward descriptive categories rather than a formal grading system, for example: in vitro; ex vivo; rodent/preclinical; livestock pilot; controlled livestock feeding trial; field/commercial evidence. This would greatly improve the critical value of the review and help readers distinguish proof of formulation from evidence of practical efficacy.
5. Please carefully audit Tables 5–7 against the cited papers and against the manuscript text. There are several apparent factual inconsistencies. For example, in the poultry section: the text identifies the hydrolysable-tannin study as being conducted in Zhongshan ducks, whereas Table 5 labels the animal category as “Chick”; conversely, the lavender-essential-oil study is described in the text as involving chicks, whereas Table 5 labels the animal category “Duck”. There are also serious terminology problems in Table 7. The main text correctly identifies the encapsulated probiotics as Bacillus coagulans SN-8 and Saccharomyces boulardii SN-6, whereas the corresponding table entry gives “Claycorbicoccus + Brachyspira yeast”. These are not equivalent organism names and the table must be corrected.
6. Some anatomical and evidential statements in Table 5 should also be reconsidered. The first poultry example concerns intramuscular administration of an antigen formulation. The table lists the target as “lymphoid tissues such as the spleen (GALT)”. The spleen should not be described as gut-associated lymphoid tissue. Moreover, evidence of altered splenic T-cell populations following intramuscular administration does not in itself demonstrate targeted delivery to GALT. Please distinguish: administration site; observed immunological response site; proposed target tissue; and directly demonstrated localisation. This is exactly why an evidence-level column would be useful.
7. The dedicated safety/regulatory discussion remains incomplete. The new discussion of regulation and safety is welcome, particularly the acknowledgement that nanoscale systems require additional investigation of long-term in vivo behaviour, absorption, metabolism, effects on microbiota and environmental safety. Nevertheless, my previous Comment requested broader consideration. The enrofloxacin example now appropriately discusses residue depletion and withdrawal periods, which is an important improvement. However, the broader Section 5.4 remains relatively general. I recommend adding a short, clearly identifiable subsection specifically entitled something similar to “Safety, residues and regulatory considerations”. It need not be long, but it should distinguish safety for: the target animal; consumers of animal-derived foods; workers/feed manufacturers; and the environment. The authors should also distinguish biodegradable food-grade carriers from persistent or novel nanoscale materials rather than treating all targeted-delivery materials as having equivalent safety profiles.
8. The conclusion still contains claims that are stronger than the evidence reviewed. The Conclusions state that the review examines how preparation methods “perform under real feed processing conditions, across various livestock species, and at industrial scales”. However, an important conclusion of the review itself is that many systems have not been adequately assessed under realistic pelleting, extrusion, storage or large-scale commercial conditions. The conclusion should therefore say that the review assesses their potential compatibility with, or discusses available evidence concerning, feed-processing and industrial-scale conditions, rather than implying that sufficient real-world performance evidence exists. Similarly, statements describing targeted delivery as “precise, efficient, and environmentally friendly” are too categorical given the limited long-term environmental and livestock-specific evidence discussed elsewhere in the manuscript. The final conclusion should explicitly state that: evidence is uneven among species; evidence is uneven among carrier technologies; much of the mechanistic literature remains preclinical or in vitro; livestock efficacy is better established for some conventional encapsulation systems than for advanced nanocarriers; commercial-scale feed-processing validation remains limited; long-term safety and residue data remain insufficient for many materials.
9. The manuscript contains several structural and cross-referencing errors that require careful proofreading. Please correct the following:

• Section 5 is “The Application of Targeted Delivery Technology in Animal Production”, and Section 5.4 follows it, but the subsequent conclusion is again numbered “5. Conclusions and Future Viewpoints”. This should presumably be Section 6.
• Section 4.3 is entitled “In vitro fluorescence imaging”, although the section explicitly discusses in vivo fluorescence imaging in live mice. The heading should be corrected.
• On page 15, the text states that the schematic showing protection, controlled release, passive targeting and active targeting is Figure 2. This schematic is Figure 1; Figure 2 is the lutein nanoparticle-preparation diagram.
• The paragraph describing the limitations of static in vitro culture systems is repeated almost verbatim twice in succession in Section 4.2.
• Please verify every table and figure call after renumbering.

Minor issues

10. Terminology in the tables should be standardised

The column heading “Boundedness” in Tables 5–7 is unclear and should be replaced by “Limitations” or possibly “Limitations/evidence gaps”.

Administration terminology should also be standardised. For example, avoid mixing “Take orally”, “Feeding”, “Feed addition”, “Oral gavage” and similar expressions where a standard terminology can be used.

Suggested terms include:

• oral gavage;
• dietary supplementation;
• oral administration;
• intramuscular injection;
• in vitro incubation;
• in situ nylon-bag assay.

11. Please distinguish evidence of targeting from inference from biological outcomes. The revised dairy-cow probiotic example now appropriately states that distal-intestinal release was inferred from faecal microbiota changes rather than directly demonstrated. This is a good revision and the same evidential standard should be applied throughout the review. For example, improved villus height, altered cytokine expression or changes in faecal bacterial abundance may demonstrate a biological response, but they do not by themselves establish that a carrier reached a particular intestinal site or underwent site-specific release. Please use terms such as “suggested”, “consistent with”, or “was inferred to” whenever localisation was not directly measured.

12. Please check Figure readability again at final publication size. The figures are informative, but several contain substantial quantities of text. Even if the source files are high resolution, readability should be checked at the size at which the figures will actually appear in the journal. Figure 3 in particular contains numerous annotations in the lower 

Comments on the Quality of English Language

A further English-language and scientific-terminology edit is needed. The revision is improved, but a number of sentences remain awkward or grammatically incomplete. Examples include constructions such as “The physical state of which…” and “Because it requires relatively sophisticated equipment…”. There are also inconsistent expressions such as “targeting the specific action site”, “rumen-indefinite digestibility”, “feed-to-meat ratio”, “glycerosugar modified”, and “sulfurized alginate”, some of which may reflect translation rather than accepted terminology. Please undertake a full scientific-language revision rather than correcting these individually.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have revised the previously mentioned comments and the manuscript now in appropriate form

Comments on the Quality of English Language

 The English could be improved to more clearly express the research.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf