Fatty Acid Composition and Aromatic Profile of Krškopolje and Modern Pig Breeds Reared Under Organic and Conventional Systems

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. Abstract needs improvement. Add a few line on methodology.
2. Line 40: explain the context of
3. Line 74-77: Diet is a key determinant of IMF; for example, diets low in amino acids can enhance IMF without promoting excessive fat deposition, a consideration particularly relevant in organic production systems where synthetic amino acids and genetically modified feed ingredients are restricted. -> Need rephrasing; start like, Diet low in amino acids can … are restricted, thus, making diet a crucial factor of IMF.
4. Sample size should be same for each breed and rearing system. This difference can impact the authenticity of the result. Please justify it and discuss the impact in discussion and this can come in the limitation.
5. 2, line 106: Explain what GC-FID and its purpose in one or two lines under the fatty acid analysis section is.
6. Line 143 and 144: write formula in equation format.
7. 3, Line 152 and 153: Two groups underwent VOC analysis; the first being representative (10 Krškopolje), and the second with proportionally more samples (21 Modern). However, there is not much information regarding how the representatives were selected and if they are actually a good representation of the larger fatty acid population. All samples should be analyzed. Furthermore, explain what is HS-SPME/GCMS and its purpose in one or two lines.
8. The study's authors state that the Krškopolje meat has higher AI and TI due to a higher SFA than expected for the meat but still remains within or close to recommended limits. More of a comprehensive discussion regarding the subjects' "healthfulness" would be useful since Krškopolje meat is conventionally viewed as "high quality." The higher SFA and TI can be contradictory to consumers interested in healthful fats. The dialogue regarding this "conflict" between sensory quality as an SFAs/MUFAs and nutritional regulations or guidelines would improve the overall quality of the paper.
9. The n-6:n-3 ratio in both breeds was >15, far exceeding the recommended limit of <4. The authors should discuss why these ratios are so high in this specific Slovenian context. Is it a result of the local feed composition (e.g., high corn/soy usage)? Suggesting specific dietary interventions (like flaxseed supplementation) to bring these ratios closer to the <4 target would increase the paper’s practical application for farmers.

 

Author Response

See attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigates the effects of breed (Krškopolje vs. modern types) and production system (organic vs. conventional) on the fatty acid profile and VOCs in pork. The topic is relevant for the journal Foods, particularly in the context of sustainable production, autochthonous breeds, and meat quality.
The study is methodologically correctly conducted (GC-FID, HS-SPME/GC-MS, multivariate analysis), and the results are consistent and logically interpreted. However, there are certain methodological and interpretative shortcomings that should be clarified.

Comments:

- In the VOC analysis, Krškopolje: n = 10 (all ORG), Modern breed: n = 21 (all CON). This practically means that the comparison of VOC profiles actually reflects breed + production system together, rather than a pure breed difference. Authors should clearly emphasize this limitation in the Discussion section, avoid formulations that imply a purely breed-related difference, and explicitly state that the VOC analysis was performed on a subset of samples from only one production system per breed. This is important for the interpretation of the multivariate analysis.

- For FA analysis, Krškopolje ORG: 36, Krškopolje CON: 35, Modern ORG: 4, Modern CON: 70. The Modern ORG group (n = 4) is extremely small. Authors should explain why this group is so small, discuss the statistical power for this subgroup, and state that results related to MB_ORG should be interpreted with caution.

- OPLS-DA validation of the FA model: R² = 0.380, Q² = 0.282, Accuracy = 82.8%. Q² < 0.3 indicates relatively weak predictive ability of the model. Authors should present permutation test results (if performed), more clearly comment on the limited separation, and avoid formulations such as “model effectively distinguished breeds” – the separation is partial.

- The study has a 2×2 design (breed × system), but the Kruskal-Wallis test was used. Authors should explain why a two-way ANOVA or GLM was not applied, whether the breed × system interaction was tested, and whether data normality was assessed. This is particularly important because the manuscript mentions an “interaction between breed and rearing system,” but a formal interaction analysis is not presented.

- Sampling period – samples were collected in 2022, 2024, and 2025. It should be stated whether seasonality was considered, whether feeding formulations were the same across years, and the possible effect of year should be briefly discussed.
- VOCs are expressed as relative percentages. It is suggested that authors more clearly state that this is a semi-quantitative analysis and avoid formulations implying absolute concentration.
- Although AI values are below 0.5, the n-6:n-3 ratio is very high (>15). The tone in the section implying nutritional superiority should be slightly moderated. It should be clearly emphasized that both profiles are far from the recommended ratio (<4).
- Terminological and stylistic corrections: “higher PUFA for 39.8%” – “higher by 39.8%”, “similar value” – “similar values”, and several minor grammatical corrections in the Introduction and Discussion sections.

Author Response

See attachement.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Below is a focused, critical review addressed to the authors, written directly and with short, targeted questions, as requested.

First, the manuscript shows a similarity index above 30%, which is too high for a research article and raises concerns about originality. Which sections are responsible for this overlap, and how will you rewrite them to ensure genuine originality, especially in the Introduction and Discussion? Please clarify whether parts of the text were reused from your previous publications and, if so, how you will substantially rephrase and refocus them.

Why is the sample size for VOC analysis so unbalanced (Krškopolje n=10 vs. modern n=21), and why are VOCs analysed only for one production system per breed? How can you justify drawing breed-level conclusions when the rearing system is confounded with breed in the VOC dataset? Please explain why this design does not bias the interpretation.

What is the rationale for pooling samples from different years (2022, 2024, 2025) without explicitly modelling “year” as a factor? How did you verify that storage time, year-to-year variability, or batch effects did not influence fatty acid and VOC profiles?

Why were only four organic samples available for modern breeds in the fatty acid analysis, and how does this low number affect statistical power and the robustness of comparisons? Have you considered reporting effect sizes and confidence intervals instead of relying mainly on p-values?

In the OPLS-DA models, Q2 values are modest (e.g., ~0.28 for fatty acids), yet you report good discrimination. How do you address the risk of overfitting, especially with relatively small and unbalanced groups? Were permutation tests or external validation performed, and if not, why?

Accuracy is reported as 100% for the VOC-based OPLS-DA. With only 31 samples, how was this accuracy calculated, and was it cross-validated properly? Please provide details on the validation procedure and clarify whether this result might be optimistic.

Several differences in fatty acids are statistically significant but numerically small. Which of these differences are biologically or nutritionally meaningful, and which are likely trivial? Can you explicitly discuss practical relevance rather than only statistical significance?

You frequently attribute differences to “genetics” or “diet,” but the actual feed composition is not described in detail. Why are precise diet formulations, fatty acid profiles of feeds, or feeding regimes not reported? How can the reader separate genetic from nutritional effects without this information?

In Table 2, many VOCs are reported as “nd” in one group and present in the other. Were detection limits established and reported? How do you ensure that “nd” truly reflects absence and not analytical sensitivity issues?

Why were VOC results analysed independently of the rearing system, while fatty acids were analysed with breed × system interactions? Given that production system clearly affects lipid composition, why was a similar multifactorial approach not applied to VOCs?

The discussion often restates results with literature support but rarely challenges your own findings. What are the main limitations of your study design, and how might they change the conclusions? Please add a more critical limitations paragraph.

Some conclusions appear stronger than the data allow, especially regarding “breed-specific markers” and “authentication.” What additional validation would be needed before proposing these compounds as reliable markers in practice?

Finally, please streamline the Discussion to reduce repetition with the Results and rewrite the most overlapping sections to address the high similarity index (>30%). The manuscript would benefit from a clearer separation between data description, interpretation, and broader implications.

Author Response

See attachement.

Author Response File: Author Response.pdf