Review Reports

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find detailed responses below and the corresponding revisions/corrections in the corrected manuscript.

Point-by-point response to Comments and Suggestions for Authors

Comments 1: The manuscript does not clearly articulate what new ecological mechanisms or management insights emerge from the findings, which weakens the impact.

Response 1: This study highlights several critical disadvantages associated with the release of hatchery-reared S. trutta juveniles into natural riverine ecosystems. Our results demonstrate that, during their first two years in the wild, hatchery-reared juveniles exhibit significantly slower growth rates compared to their wild-born counterparts. Furthermore, a high prevalence of fin erosion was observed among reared specimens, a condition that persisted throughout the study period.

Our haematological analysis provides novel insights by evaluating physiological markers—such as glucose levels—under natural conditions. These findings demonstrate that physiological indicators do not always directly correlate with growth or overall fitness outcomes. Instead, our results indicate that both environmental variability and rearing origin interact to shape the physiological profile of the fish, with these effects being strongly modulated by stream-specific conditions. Consequently, we emphasise that haematological responses must be interpreted within a broader ecological framework.

Overall, these findings provide a comprehensive assessment of the ecological mechanisms and management implications of current stocking practices. By clarifying the interplay between hatchery-origin, environmental adaptation, and physiological health, this study offers a robust basis for refining salmonid management and conservation strategies in Lithuania

Comments 2: A fundamental limitation of the study is the lack of true experimental control between streams. The authors should discuss this limitation more explicitly and avoid overstating causal conclusions.

Response 2: The primary objective of this study was to evaluate and compare the growth and physiological condition of wild and hatchery-reared S. trutta juveniles during their first two years of life, preceding smoltification and seaward migration. Two distinct tributaries were intentionally selected to determine whether the observed differences between hatchery-reared and wild cohorts remained consistent across varying environmental contexts. While this study design did not initially aim to conduct a direct comparative analysis between the two streams—and thus did not strictly adhere to a controlled experimental framework for stream comparison—the “stream” factor was subsequently integrated into our statistical models. This inclusion was essential to account for site-specific environmental variance and to ensure that the growth trajectories of fish from distinct habitats were appropriately partitioned in the analysis. Consequently, the wild individuals served as the definitive control group against which the performance of the hatchery-reared juveniles was assessed under natural conditions.

Comments 3: The authors state that hatchery fish originated from a single breeding pair to minimize genetic variation. While this approach standardizes genetics within the hatchery group, it introduces a serious genetic bottleneck. Using offspring from only one pair may lead to reduced genetic diversity, inbreeding effects, and lower adaptive potential. These genetic constraints could partly explain the lower performance of hatchery fish, yet the manuscript does not sufficiently discuss this issue. A stronger discussion on genetic limitations of hatchery programs is required.

Response 3: This study spanned a four-year period (2019–2022), during which a total of 15,000 S. trutta juveniles were stocked. Throughout the investigation, the stocked material was derived from four distinct parental pairs (eight individual specimens in total), with each annual cohort originating from a single breeding pair. This experimental design was primarily adopted to mitigate the ecological impact of removing reproductive adults from the wild and to optimise the efficiency of parental sampling and handling during the artificial breeding process. A single breeding pair proved sufficient to meet the annual requirement of approximately 4,000 juveniles for stocking (as detailed in the Methods section, lines 124–127). However, it is acknowledged that the limited number of parental individuals used in this study may have constrained the genetic diversity of the hatchery-reared fry, potentially reducing their adaptive capacity. These genetic limitations and their implications for the study's findings are further addressed in the Discussion section (lines 550–557).

Comments 4: The growth analysis indicates that hatchery fish were initially larger and wild fish caught up in size by age 1+. However, the authors interpret this as evidence of slower growth of stocked fish. This interpretation may be misleading. The observed convergence could also result from: a) compensatory growth in wild fish; b) density-dependent effects, c) environmental adaptation differences. More cautious interpretation is needed.

Response 4: he growth dynamics of wild and hatchery-reared S. trutta juveniles were compared across two distinct streams—characterised by differing thermal regimes, fish assemblages, and population densities—and across four independent annual cohorts. Notably, the pattern of slower growth in reared juveniles relative to their wild counterparts was consistently observed in both river systems and throughout all studied cohorts. This recurrence strongly suggests that the reduced growth rate of artificially reared juveniles is a robust phenomenon under natural conditions. Since both groups (wild and reared) were subjected to identical environmental pressures and resource availability within each respective stream, the observed disparities are unlikely to be an artefact of density-dependent effects. Instead, these findings point toward maladaptation issues unique to the stocked hatchery-reared individuals. Our results align with recent literature indicating that hatchery-reared salmonids frequently exhibit altered growth trajectories and diminished ecological performance upon introduction to natural environments.

Comments 5: The high frequency of fin damage in hatchery fish is an interesting finding. However, the manuscript assumes that this damage is primarily related to hatchery conditions. While this is plausible, the authors should acknowledge that fin damage may also result from environmental stress, predation attempts, or aggressive interactions after stocking. Furthermore, the functional consequences of fin damage (e.g., reduced swimming performance) are speculative and not tested in this study.

Response: 5. We have significantly increased discussion on the reasons for fin damage appearance (Lines 493-500) and expand discussion on the association of fin damages with the reduced fish growth covered it by several citations (Lines 500-502 and 510-513).

Comment 6: The hematological section is informative but somewhat superficial. Important issues include: 1) The physiological meaning of MCV variation between streams is not clearly explained; 2) The interpretation of glucose elevation in hatchery fish remains speculative; 3) Stress biomarkers such as cortisol were not measured. Additionally, the manuscript should clarify whether fish were fasted or handled prior to blood sampling, as these factors can significantly influence glucose levels.

Response 6: We thank the reviewer for these helpful comments and have revised the manuscript accordingly. The physiological interpretation of MCV has been clarified by linking erythrocyte size to oxygen transport capacity and metabolic activity, and by relating inter-stream differences to environmental variability. The interpretation of elevated glucose levels has been revised to avoid overstatement. Cortisol was not measured in this study, as the primary aim was to assess growth and general physiological condition of juveniles under natural field conditions rather than to quantify acute stress responses. Moreover, cortisol is known to be highly sensitive to capture and handling, which can lead to rapid and transient increases that are difficult to standardize under field sampling conditions. Therefore, we focused on haematological parameters that are less immediately affected by short-term disturbance. We acknowledge that inclusion of cortisol measurements would provide additional insight and highlight this as a direction for future research. Finally, the methodology has been clarified to indicate that fish were sampled directly from the natural environment and blood was collected immediately after capture.

Comment 7: The gut microbiota analysis is the weakest part of the study. Limitations include: 1) Very small sample size (n = 40 fish); 2) Pooling of samples (3–4 individuals); 3) which reduces biological resolution; 4) Only cultivable bacteria were analyzed; 5) No identification of bacterial taxa. Given the current standards in fish microbiome research, culture-based enumeration provides limited ecological insight. High-throughput sequencing approaches would have been more informative. The authors should either significantly expand this section or clearly acknowledge its limitations.

Response 7: Using 5–10 fish per experimental group is a common and recommended practice in studies of fish intestinal microbiology. It helps ensure a reliable representation of intestinal microbial communities and addresses animal welfare by avoiding unnecessary killing. In our study, we randomly selected ten fish for each experimental group. For example, in salmonid research cited in our study, gut microbiome analysis was conducted on eight individual fish per experimental group (Uren Webster et al., 2020).

Pooling fish gut content samples is a common technique in microbiological and molecular studies to increase population-level coverage and manage small sample sizes.  The aim of our study was to reduce stochastic variation among individual fish and obtain a general overview of the population. Additionally, in both molecular and classical microbiological studies, intestinal contents are pooled when the fish individuals are very small, and the gut contents from a single individual do not exceed 100 mg. In our study, the gut contents of one fish weighed less than 100 mg. Therefore, by pooling the gut contents of several fish, we obtained 300 mg, and this approach allowed more accurate, methodical counts of viable bacteria. For example, fish gut metagenomic studies require a 500 mg sample.

Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the “gold standard” for bacterial quantification and measuring viability across various disciplines (Meyer et al., 2023). The disadvantage of the molecular approach (PCR and sequencing) is that it only detects the presence of bacterial DNA but does not indicate bacterial viability (Hartley et al., 2019). Only viable gut bacteria perform metabolic, immunological, and protective functions and are crucial for the animal's health and functional state (Butt and Volkoff, 2019). Assessing viable gut bacteria, rather than just their presence, offers a more accurate understanding of the functional gut microbiome.

We also agree that this is not a complete and comprehensive study of the gut microbiota (no identification of bacterial taxa), but our results provide additional details on the differences between wild and artificially reared Salmo trutta fry after stocking.

References used for this answer:

Meyer, C.T., Lynch, G.K., Stamo, D.F., et al. (2023). A high-throughput and low-waste viability assay for microbes. Nature Microbiology, 8, 2304–2314. https://doi.org/10.1038/s41564-023-01513-9

Hartley, M.G., Ralph, E., Norville, I.H., Prior, J.L., & Atkins, T.P. (2019). Comparison of PCR and viable count as a method for enumeration of bacteria in an A/J mouse aerosol model of Q fever. Frontiers in Microbiology, 10, 1552. https://doi.org/10.3389/fmicb.2019.01552

Butt, R.L.; Volkoff, H. Gut Microbiota and Energy Homeostasis in Fish. (2019). Frontiers in Endocrinology, 10, 9. https://doi.org/10.3389/fendo.2019.00009

Comment 8: The manuscript uses linear mixed models and generalized linear models, which are appropriate. However, several statistical details are unclear: 1) Model assumptions are not fully reported; 2) Effect sizes are not presented; 3) Confidence intervals are missing; 4) Sample sizes for some comparisons are relatively small. Providing effect size estimates and graphical uncertainty measures would improve transparency.

Response 8: Unfortunately, reporting on assumption testing for all the models used would result in reduced readability of the paper. Instead, we attempted to be as transparent as possible by depicting the data points and their distribution on the original scale in plots of models to which the normality and homoscedasticity assumptions apply. These plots serve as proof in cases where we had (or did not have to) log-transform certain variables to avoid rough violations of the assumptions. We tested multiple assumptions in R using package performance. In response, we now included a sentence on used packages and their purposes at the end of each method’s subsection.

We assume that this mostly relates to growth analyses, for which growth rate difference may be biologically relevant. In response, we now included pairwise comparisons of estimated growth slopes (log-log scale) in our analyses and along with the growth plots (Figure 4C, F).

Indeed, the CI, which were very overlapping for the 4 investigated groups and thus negatively affected plot readability (see example below), were consciously removed from the original-scale growth plots (now Fig. 4A, D). We thus chose to show the important cross sections with 95% CI as whiskers in the additional pairwise comparisons plots (now Fig. 4B, E), and now in the estimated growth slopes (now Fig. 4C, F).

We agree that more data points are always better for statistics, but the number of data points in biological science often comes at a cost to animal life. We were transparent with the numbers, and we see no evidence that our dataset was insufficient for a sound statistical analysis, and we even find it quite extensive in comparison to other fish studies.

We hope the added depiction of growth slopes with CI contributed to both aspects.

Comment 9: The manuscript contains multiple grammatical inconsistencies and awkward phrasing. Examples include: 1) “rearrests” instead of “rarest”; 2) “owes less damages counts”; 3) inconsistent use of scientific terminology. The manuscript requires professional language editing.

Response 9: Manuscript language was revised, pointed phrasing changed.

Comment 10: Several terms should be standardized: 1) "fitness" vs "body condition"; 2) "reared fish" vs "stocked fish"; 3) "necrosis" vs "fin damage". Consistency will improve readability.

Response 10: The pointed terms were standardized.

Comment 11: Some figures need improvement.

Response 11: Figure and figure captions have been improved.

Comment 12: Some references are outdated. The introduction and discussion would benefit from incorporating recent studies (2020–2024) on: 1) hatchery–wild interactions; 2) stocking effectiveness; 3) salmonid ecological adaptation.

Response 12: We thank the reviewer for this valuable suggestion. Recent literature was incorporated into both the Introduction and Discussion sections to reflect current knowledge on hatchery–wild interactions, stocking effectiveness, and ecological adaptation of salmonids.

• Added recent references and supporting text on hatchery–wild interactions, including McMillan et al. (2023), Harrison et al. (2026) and Fabiano and Harrison (2025), (Introduction, page 2):
• Expanded interpretation of growth differences and incorporated recent literature on ecological performance and stocking outcomes (McMillan et al., 2023; Harrison et al., 2024; Fabiano and Harrison, 2025; Reiser et al., 2026), (Discussion, page 16):

Added reference to recent studies on pathogen-related mechanisms affecting fish performance (Krkošek et al., 2024). (Discussion, page 15).

Comment 13: Recommendations for improvement

The manuscript would benefit from:

1. Stronger discussion of experimental limitations.
2. Expanded ecological interpretation of results.
3. Improved statistical reporting.
4. Revision of the microbiota section.
5. Professional English editing.
6. More cautious conclusions regarding stocking effectiveness.

Response 13: All these recommendations were carefully taken into consideration and each of them separately were acknowledged. All changes that have been made are already mentioned in the point-by-point comments reply above.

Response to reviewer 2 comments

Point-by-point response to Comments and Suggestions for Authors

Comment 1: The full name of ICES needs to be defined when it first appears in the form of abbreviation.

Response 1: The full name of ICES was defined (Line 83).

Comment 2: The authors need to further clarify the global status of Salmo trutta population conservation in the Baltic Sea region of Lithuania on a global scale, as well as the current research progress and main problems in this regard. 

Response 2: Global status of Salmo trutta populations as well as its status in the Baltic Sea region and Lithuania was clarified (lines 49-59).

Comment 3: Detailed description is required for the facilities and equipment used for artificial fry cultivation, feed of fry and juvenile fish, methods of artificial fry cultivation, and feeding management, which helps to gain a deeper understanding of the underlying reasons for the differences.

Response 3: We thank the reviewer for this important and constructive comment. We agree that a more detailed description of hatchery rearing conditions improves the transparency and reproducibility of the study. Therefore, we have expanded the Methods section to include quantitative information on rearing environment, including water temperature, oxygen concentration, stocking densities, and feeding regime. Specifically, we clarified that fish were reared under controlled hatchery conditions using flow-through systems, with water temperature maintained within optimal ranges for salmonid development, dissolved oxygen above 6 mg/L, and densities adjusted according to developmental stage. Feeding consisted of live feed during early ontogeny followed by a transition to commercial starter diets. We also clarified that fish were held under controlled conditions and released into streams after a short acclimation period, minimizing stress and preventing escape during rearing. In addition, we clarified that wild S. trutta juveniles originated from natural reproduction within the studied streams and were sampled from the same river sections as stocked individuals, ensuring comparability between groups.

These additions improve the methodological clarity and strengthen the interpretation of the results (page 4, lines 127 – 137).

Comment 4: Authors need to describe in detail how to contain the fry from escaping during the rearing periods.

Response 4: Artificial bred fry during the rearing periods were kept indoors, in the closed tanks. There was no way to escape. After initial rearing the fry was marked and released to wild nature.

Comment 5: Authors need to clarify how wild fry were obtained.

Response 5: Fish sampling was performed using battery-powered electric fishing gear. All fish sampling methods were explained in the methods section (lines 184-185).

Comment 6: It is necessary to clarify whether the age of wild fry is similar to that of farmed fry. 

Response 6: The size and age of sampled wild and reared S. trutta specimens were identical. This information is provided in the methods section (tables 3, 4).

Comment 7: Table 3 shows the data for 4 consecutive years (2019, 2020, 2021, and 2022). Phenotypic indicators determined were presented with only three years (2019, 2020 and 2022). This needs to be explained clearly. 

Response 7: Yes indeed, fish (1348 individuals) for the growth analyses were sampled in 2019-2022 years. Fish (699) for fin damage was sampled in 2020-2022 years. Fish (456 ind.) for haematological assessment was sampled in 2019, 2020 and 2022. Fish (40 ind.) for gut bacteria counts were sampled only in 2019. Significant subsamples of the overall collected fish were used for fins damage, blood or gut bacteria assessments. This information is clearly presented in methods section.

Comment 8: Tables 6-11: Data of phenotypic indicators were subjected to ANOVA analysis in response to factors (time, origin and stream). Therefore, specific statistical methods are needed to evaluate these effects: the effects of factors (time, origin and/or stream) on phenotypic indicators. 

Response 8: We are not sure if we really understood the underlying remark, but we agree that biological effect sizes were not clearly provided where they were indeed relevant - which is in the growth comparison. Thus, growth slope comparisons and their pairwise comparisons were now included in Fig. 4. We would argue that effect sizes in all other analyses are not standard (or very meaningful) to provide, as they are anyway clearly depicted in variation by factor plots and could be more or less self-derived by the reader in need.

Comment 9: Figure 4, figure 5 and figure 6 lack self-evidence, hard to understand. The expression of "reared vs wild" in the text and figures is inconsistent.

Response 9: We attempted to clarify these figure plates by adding letter indices for each part, including extra explanations in the figure captions, and generally improving the visual aspect of the figures (labeling, font sizes, etc). The term “native” was changed to “wild” throughout the paper and figures to maintain consistency.

Comment 10: The author's analysis of the reasons why the degree and frequency of fin damage in farmed fish are significantly higher than those in wild fish is too open and not convincing. 

Response 10: We have improved discussion on the fins damage reasons and higher prevalence in reared fish in the discussion part (lines 488-513).

Comment 11: About discussion on body fitness with haematological parameters, all relevant analyses are off topic and argumentation on the impact of one specific factor on blood components. This requires a deep correlation analysis between the changes of haematological parameters and specific rearing conditions (feed, stocking density, management, etc.). 

Response 11: We thank the reviewer for this important comment. We agree that establishing direct causal relationships between specific rearing conditions (e.g., feed, stocking density, management) and haematological parameters would require a controlled experimental design and targeted correlation analysis.

However, the aim of the present study was not to isolate the effects of individual rearing factors, but rather to compare the physiological responses of wild and hatchery-origin juveniles under natural environmental conditions.

Comment 12: The gut bacteria detection was conducted during a relatively short stocking period in the natural environment, compared to a long-term stocking period from fry to smolt stage. The detection may not explain the differences between farmed and wild fish from the perspective of gut microbiota. Furthermore, changes in the gut microbiota cannot give a clear conclusion on which factor could determine the difference in growth between reared and wild fish. 

Response 12: Considering your comments, we have revised and rewritten the discussion section. (Lines: 634- 657.)

Comment 13: Conclusions need to be adjusted accordingly after re-analysis and discussion.

Response 13: Conclusions were adjusted accordingly after MS review.