Review Reports - Amino Acid Composition of Meat from Smena 9 Broiler Chickens During Ontogeny Under a High Stocking Density and Dietary Adaptogens Complex

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title

The title is too lengthy and needs revision

Abstract

Need to add birds per treatment,

Avoid use of words like partial compensation.

Introduction

The author did not mention the reason for using these dosage levels of Vitamin C and DHQEC.

Materials and Methods

The inclusion level of Wheat is too high, which may negatively influence the birds. Do the authors add any enzyme to improve its digestibility?

The authors do not mention any specific details about number of birds placed in each stocking density and how many birds are placed when stocking density increased by 10%.

Similalry, the division of birds in treatments and replicates also not mentioned in this study.

The authors repeatedly used the word "stocking density stress"; however, no direct physiological stress indicators were measured in the current study that can establish that either the birds were in stress or not.

Results

It is suggested to remove the comparison of aminoacid made on 24 days of age, as the 4th treatmen started on 21st day of age. 03 days are very short time to make changes in aminoacid concentration.

Describe abbreviations of Table 8 in footnotes

Discussion

The authors related heat stress with stocking density stress, however, both have different mechanisms, it is better to avoid unnecesaary correlations.

The authors focused more towards mechanisms after change of amino acid and a little explanation is present regarding why the concentration changed.

"Somewhat higher" is not a scientific term. If the results are non-significant, it is suggest not to make thing interesting artificially.

Author Response

Dear Reviewer,

We are grateful for your thorough analysis of our work and for the comments you provided, which have improved our manuscript. We have carefully considered and incorporated all of your suggestions.

Changes made to the manuscript are highlighted in blue, while changes made to improve the quality of the scientific English are highlighted in red. In addition, the reference list has expanded, with the additions highlighted in red. The citation order of the references has also updated accordingly.

The title is too lengthy and needs revision

Need to add birds per treatment, Avoid use of words like partial compensation.

We agree with the reviewer's opinion that it is necessary to specify the number of individuals per 1 m².

We have added the following text to the abstract:

The planting density changed as the bird grew. On day 21, the bird planting density in the S(-)CON group was 38 heads per 1 m², in the other groups it was increased by 10% and amounted to 42 heads per 1 m². Each week, the planting density changed and amounted to 32 and 35 heads per 1 m² in 28-35 days, 21 and 23 heads per 1 m² in 35-42 days, and 17 and 19 heads per 1 m² in 43 days and until the end of fattening, respectively.

The author did not mention the reason for using these dosage levels of Vitamin C and DHQEC.

The mechanism of the synergistic action of biologically active substances on the organism of animals and poultry is well documented. This primarily concerns antioxidants, which, when combined in diets, perform a protective function against oxidative cell damage. Vitamin dosages were calculated based on an analysis of literature sources and established norms for their use in poultry nutrition, taking into account their synergistic effects. We selected 30% of the daily requirement for each vitamin, considering the multidirectional action of activating the body’s antioxidant defense and immunostimulation. The dosage of dihydroquercetin (DHQ) was tested in previously conducted studies on poultry and monogastric animals (Ostrenko K.S., Nekrasov R.V., Chabaev M.G. et al. Effect of feed additive DHQEC on antioxidant status of fattening pigs (in Russian). Sib. J. Life Sci. Agric, 2023, 15(6), 222–245. https://doi.org/10.12731/2658-6649-2023-15-6-966; Semenova A.A., Kuznetsova T.G., Pchelkina V.A. et al. Muscle tissue microstructure in hybrid pigs (Sus scrofa domesticus L.) under intensive fattening influenced by adaptogens (in Russian). Sel’skokhozyaistvennaya Biologiya, 2023, 58(2), 355–372. https://doi.org/10.15389/agrobiology.2023.2.355eng; Bogolyubova N.V., Nekrasov R.V., Lakhonin P.D., Kolesnik N.S., V’yuchnaya P.S., Zelenchenkova A.A., Nikanova D.A., Bogolyubova Yu.A. Biological and productive effect of a complex of adaptogens on broiler chickens (Gallus gallus L.) of Smena 9 cross under increased stocking density (in Russian). Sel’skokhozyaistvennaya Biologiya, 2026, 61(2), 304–319. https://doi.org/10.15389/agrobiology.2026.2.304rus). Furthermore, were also taken into account the results of earlier studies (Fomichev Yu.P. Flavonoid-dihydroquercetin in human and animal nutrition, safety of agricultural products. Efficient Animal Husbandry, 2018, 4(143):58-60).

Materials and Methods. The inclusion level of Wheat is too high, which may negatively influence the birds. Do the authors add any enzyme to improve its digestibility?

The finishing compound feed indeed contains 55% wheat. At the final stage of broiler fattening, this strategy contributes to improved carcass quality and reduced feed costs. The compound feed used included a premix, which in the finishing feed contained the necessary amounts of enzymes to aid in the digestion of feed nutrients. These enzymes include phytase, non-starch polysaccharidases (xylanase, beta-glucanase), and proteases. Thus, the composition of the compound feed and premix met the physiological requirements for nutrients and biologically active substances.

The authors do not mention any specific details about number of birds placed in each stocking density and how many birds are placed when stocking density increased by 10%. Similalry, the division of birds in treatments and replicates also not mentioned in this study

This information has incorporated into the abstract, the materials and methods section, and is presented in Table 3.

The authors repeatedly used the word "stocking density stress"; however, no direct physiological stress indicators were measured in the current study that can establish that either the birds were in stress or not.

We are grateful to the reviewer for this question. The effect of stocking density on blood biochemical parameters, antioxidant status, and hormonal status was reflected in our recent publication (Bogolyubova N.V., Nekrasov R.V., Lakhonin P.D., Kolesnik N.S., V'yuchnaya P.S., Zelenchenkova A.A., Nikanova D.A., Bogolyubova Yu.A. Biological and productive effect of a complex of adaptogens on broiler chickens (Gallus gallus L.) of Smena 9 cross under increased stocking density. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2026, 61(2), 304–319. https://doi.org/10.15389/agrobiology.2026.2.304rus). In this study, changes in the level of cortisol and the activity of antioxidant enzymes in the blood had the character of trends (p>0.05), which does not allow us to assert the presence of systemic stress with complete statistical certainty. We admit that the use of the term "stress" as an established fact in the current manuscript was unnecessarily categorical. However, the same parallel study revealed statistically significant changes in key biochemical parameters consistent with stress-induced metabolic alterations. By day 34, birds in the high-stocking-density group showed a significant increase in glucose (+4.1%, p = 0.005) and total bilirubin (+31.7%, p = 0.02). By day 52, we observed a significant increase in total protein (+4.5%, p = 0.001) and glucose (+4.4%, p = 0.006), along with a decrease in albumin (-1.5%, p < 0.001) and AST activity (-26.4%, p = 0.007), as well as an increase in triglycerides (+5.8%, p = 0.005)( The influence of simulated stress on the biochemical parameters of broiler chickens / P. D. Lakhonin, N. V. Bogolyubova, R. V. Nekrasov [et al.] // Veterinary medicine and feeding. – 2025. – No. 6. – pp. 62-67. – DOI 10.30917/TT-VK-1814-9588-2025-6-14). These changes—hyperglycemia, hyperbilirubinemia, dysproteinemia, and elevated triglycerides—are well-documented indicators of systemic stress, reflecting glucocorticoid-mediated gluconeogenesis, oxidative damage, inflammatory response, and lipid mobilization in poultry subjected to adverse environmental conditions.But the results of our other work (Expression of antioxidant protection and immunity genes in the tissues of broiler chickens under the influence of a complex of adaptogens / P. S. Vyuchnaya, P. D. Lakhonin, N. V. Bardukov, N. V. Bogolyubova // International Bulletin of Veterinary Medicine. – 2025. – No. 2. – pp. 176-189. – DOI 10.52419/issn2072-2419.2025.2.176.) the molecular markers of stress were evaluated and a statistically significant suppression of the expression of the immunity and antioxidant protection genes Nrf2, GSH-Gpx and HO-1 in the intestinal and liver tissues of broiler chickens was shown. A decrease in the activity of key antioxidant enzymes (Nrf2, GSH-Gpx, HO-1) is a generally recognized indicator of oxidative stress induced by chronic exposure to adverse factors. Oxidative stress is considered as one of the main mechanisms of the pathogenesis of stress reactions in birds. The positive correlation we found (r = 0.983, p < 0.05) between AvBD9 expression in the caecum and average daily growth confirms that the suppression of protective mechanisms we observed is associated with decreased productivity, a classic integral indicator of chronic stress in animals and poultry.

We have made some edits to the text of the article— we have removed categorical statements about stress, everywhere we write "high-density exposure" or "stress factor".

Also we added text in the introduction.

It is suggested to remove the comparison of aminoacid made on 24 days of age, as the 4th treatmen started on 21st day of age. 03 days are very short time to make changes in aminoacid concentration.

We acknowledge the reviewer's concern. Nevertheless, the sampling scheme was predetermined by the experimental design. Furthermore, the analysis of age-related dynamics of amino acid content constituted one of the primary objectives of the study. Consequently, we consider it appropriate to keep these data in the manuscript

Describe abbreviations of Table 8 in footnotes

We would like to note that the abbreviations are listed in Table 5, and these same abbreviations are used consistently across all following tables. In response to the reviewer's comment, we have also added the abbreviations to Table 8.

The authors related heat stress with stocking density stress, however, both have different mechanisms, it is better to avoid unnecesaary correlations.

High stocking density is problematic not only because it restricts spatial movement but primarily because it disrupts heat exchange, thereby inducing heat stress. Consequently, stocking density is intimately linked to heat stress. Moreover, evidence accumulated over the past two decades has unequivocally shown that the majority of stresses, irrespective of their source, are associated with an imbalance between free radical production and detoxification (Surai P.F., Kochish I.I., Fisinin V.I., Kidd M.T. Antioxidant defence systems and oxidative stress in poultry biology: an update. Antioxidants, 2019, 8(7): E235. https://doi.org/10.3390/antiox8070235)."

The authors focused more towards mechanisms after change of amino acid and a little explanation is present regarding why the concentration changed.

We agree with the reviewer's comment and have added to the discussion a part about why the amino acid composition of poultry meat may change. In response to the reviewer's suggestion, we have supplemented the Discussion with a mechanistic rationale for the observed shifts in muscle amino acid profiles. The concentration of free and protein-bound amino acids in muscle tissue reflects a dynamic equilibrium between exogenous supply, endogenous synthesis, and catabolic losses. Our previous findings indicate that dietary adaptogens improved antioxidant defense mechanisms, thereby mitigating oxidative damage to muscle proteins and sparing sulfur-containing amino acids (particularly methionine and cysteine) from excessive utilization in glutathione biosynthesis. At the same time, enhanced immune competence and improved proteolytic digestibility of the diet (Bogolyubova et al., 2026) promoted a greater postprandial amino acid flux into the portal circulation. Consequently, the net change in individual amino acid concentrations in muscle tissue can be attributed to the synergistic action of augmented intestinal uptake and diminished oxidative degradation, rather than to dietary intake alone.

"Somewhat higher" is not a scientific term. If the results are non-significant, it is suggest not to make thing interesting artificially.

We agree with the reviewer's comment and have incorporated it into our manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript provided to me for review examines the amino acid composition of meat from Smena 9 broilers fed a normal diet and a diet enriched with dihydroquercetin and vitamins E and C. My main remarks are related to the design of the experiment and the statistical processing of the results. The authors use a positive and negative control, which differ only in poultry stocking density, and 2 groups fed the enriched diet – one from the first day, and the other from the 21st day. In principle, such a grouping is possible, but when comparing the results between the groups in this situation, it will not be possible to state whether the differences in amino acid composition are due to the diet or to the stocking density. It is more correct to make a comparison only between the groups with the same stocking density to study the influence of the diet, and if desired, a second comparison only between the two control groups to study the influence of the stocking density. The broilers were slaughtered on the 24th, 34th and 52nd days. The authors claim that 10 of each group were slaughtered at each age. This makes 4 groups × 3 ages × 10 broilers – a total of 120 broilers. While in the abstract (line 37) and in Material and Methods (line 123) you claim that you used 160 broilers in the experiment. Please explain where this difference comes from or correct the number of broilers used. In material and methods you claim that a three-factor analysis of variance was performed, obviously referring to the factors of slaughter age, group factor (diet+stocking density) and gender. However, nowhere is the interaction between the studied factors shown. This is permissible only if in all cases the reliability p>0.05 and even then this should be noted. Otherwise, it is appropriate to include the interactions, although then the interpretation of the obtained results will probably change. If you have not studied the interaction between the factors, I would recommend that you change your statement and describe that a factorial ANOVA with main effects only was performed. In tables 8, 9 and 10 you give Fisher criterion values at p=0.05, which in my opinion is unnecessary, since the “p” values are a direct consequence of the F values. In many places in the tables, when there is reliability (p<0.05), the differences between the individual groups according to Tukey’s test are not shown (TYR and SNEAA in table 5; ASP, HIS and SFAA at breast meat, ASP and THR at thigh muscle in table 6 and other). Please make the necessary corrections.

Author Response

Dear Reviewer,

We are grateful for your thorough analysis of our work and for the comments you provided, which have improved our manuscript. We have carefully considered and incorporated all of your suggestions.

It is more correct to make a comparison only between the groups with the same stocking density to study the influence of the diet, and if desired, a second comparison only between the two control groups to study the influence of the stocking density

We agree with the reviewers that it is better to compare groups with the same stocking density. In our research, we conducted a comparative analysis between all the groups studied, including the S(-)CON and S(+)CON groups, as well as the S(+)CON and S(+)DHQEC_21 and S(+)DHQEC_1 groups. The main conclusions were drawn by us in relation to groups with the same stocking density.

The broilers were slaughtered on the 24th, 34th and 52nd days. The authors claim that 10 of each group were slaughtered at each age. This makes 4 groups × 3 ages × 10 broilers – a total of 120 broilers. While in the abstract (line 37) and in Material and Methods (line 123) you claim that you used 160 broilers in the experiment. Please explain where this difference comes from or correct the number of broilers used.

We completely agree with the reviewers. The fact is that 160 chickens actually participated in our experiment. At the final slaughter, muscle tissue samples were taken from 10 broilers from each group (40 heads in total). This was done in order to equalize the amount during all sampling periods. It would be more correct to specify 120 broilers. We corrected this error.

In material and methods you claim that a three-factor analysis of variance was performed, obviously referring to the factors of slaughter age, group factor (diet+stocking density) and gender. However, nowhere is the interaction between the studied factors shown. This is permissible only if in all cases the reliability p>0.05 and even then this should be noted. Otherwise, it is appropriate to include the interactions, although then the interpretation of the obtained results will probably change. If you have not studied the interaction between the factors, I would recommend that you change your statement and describe that a factorial ANOVA with main effects only was performed.

We agree with the comment of the reviewers, a multifactorial analysis of the interaction of factors was not conducted. We corrected the name of statistical analysis in Materials and Methods to factor ANOVA with main effects.

In tables 8, 9 and 10 you give Fisher criterion values at p=0.05, which in my opinion is unnecessary, since the “p” values are a direct consequence of the F values.

We agree with this remark, but for greater clarity, we would like to leave the Fischer criterion in tables.

In many places in the tables, when there is reliability (p<0.05), the differences between the individual groups according to Tukey’s test are not shown (TYR and SNEAA in table 5; ASP, HIS and SFAA at breast meat, ASP and THR at thigh muscle in table 6 and other). Please make the necessary corrections.

We agree with you that the identified reliability of changes in the value of the indicator when comparing groups is expressed in specific reliable changes between groups (usually and, first of all, extreme in value).But this is not always the case. The Tukey test controls the level of type I error (family error) in multiple comparisons. However, it can be quite conservative, which reduces the likelihood of detecting reliable differences between groups if they exist but are outside the confidence interval.

The data were tested for normal distribution using the Kolmogorov-Smirnov and Shapiro-Wilk tests and made an analysis of the data analysis of variance, and factor analysis, followed by a Tukey post-hoc test.

It is possible that another method would have shown pairwise significant differences, but we cannot rule out this possibility.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall Scientific Assessment: The research presented in this manuscript investigates the amino acid composition of breast and thigh muscles of Smena-9 broiler chickens reared under increased stocking density and supplemented with a complex of adaptogens consisting of dihydroquercetin, vitamin E, and vitamin C. The topic is relevant because intensive poultry production systems frequently expose birds to environmental stressors that may compromise growth performance, welfare, and meat quality. Furthermore, the nutritional quality of poultry meat, particularly its amino acid composition, represents an important aspect of consumer health and product value. The study has several strengths, including the evaluation of amino acid profiles in different muscle types, the consideration of age-related changes throughout ontogeny, and the investigation of a nutritional intervention intended to mitigate stress effects. However, several methodological, analytical, and interpretative limitations reduce the scientific impact of the manuscript. The most important concerns include uncertainty regarding the true experimental unit used in the statistical analyses, limited validation of the stocking-density stress model, excessive reliance on descriptive comparisons among amino acids, insufficient mechanistic explanations linking stress and amino acid metabolism, and a large number of statistical comparisons without discussion of potential inflation of Type I error. Additionally, the manuscript suffers from organizational issues, extensive and repetitive result descriptions, and numerous English language deficiencies that impair readability. Although the dataset generated is extensive and potentially valuable, substantial revisions aimed at improving methodological clarity, strengthening biological interpretation, enhancing statistical rigor, and improving scientific writing are necessary before the manuscript can be considered for publication.

Title: The title is descriptive and adequately reflects the principal variables investigated in the study, including amino acid composition, ontogenetic development, stocking density, and adaptogen supplementation. However, it is excessively long and somewhat difficult to read. In addition, the expression “correction with a complex of adaptogens” is unusual in scientific English and does not clearly communicate the nature of the intervention. The title emphasizes all experimental factors simultaneously, which reduces its focus and impact. A shorter and more precise title highlighting the effects of increased stocking density and adaptogen supplementation on amino acid composition in broiler meat would improve clarity and scientific appeal.

Abstract: The abstract provides a general overview of the objectives, experimental design, and principal findings of the study. The authors clearly describe the treatments and summarize the major observations regarding age-related changes and the effects of adaptogen supplementation. However, several shortcomings are evident. The biological rationale supporting the study is not sufficiently developed, and the knowledge gap being addressed is only briefly mentioned. The conclusions tend to be stronger than the evidence generated by the study. In particular, the authors suggest that the adaptogen complex compensates for stress-induced alterations in meat quality, yet no direct physiological, endocrine, oxidative, or welfare-related indicators were measured to confirm the occurrence of stress or the mechanisms through which the additives exerted their effects. Furthermore, numerous numerical findings are reported without prioritizing those that are biologically most meaningful. As a result, the abstract would benefit from a more critical interpretation of the findings and a clearer acknowledgment of the study’s limitations.

Introduction: The introduction provides substantial background information regarding poultry meat composition, amino acid functionality, stress physiology, and antioxidant supplementation. Nevertheless, the section is predominantly descriptive and lacks sufficient analytical depth. The physiological mechanisms through which stocking density may influence muscle growth, protein deposition, amino acid metabolism, and meat quality are discussed only superficially. Although oxidative stress is repeatedly mentioned, the manuscript does not adequately explain how oxidative damage could specifically alter amino acid composition within muscle tissue. The literature review includes studies involving amino acid profiles and antioxidant supplementation, but there is limited integration of previous findings and little discussion regarding conflicting results reported in the literature. The authors identify a lack of information regarding amino acid composition under different stocking densities; however, the scientific hypothesis remains broad and insufficiently mechanistic. Stronger synthesis of previous studies and a more explicit biological framework would substantially improve the introduction.

Materials and Methods: The experimental design combines nutritional intervention and environmental challenge in an attempt to evaluate their effects on amino acid composition in broiler meat. The description of the amino acid analytical procedures is detailed and appears technically appropriate, particularly the use of ion-exchange chromatography with post-column derivatization. However, several methodological aspects require clarification. The most important issue concerns the experimental unit used in the statistical analyses. Because stocking density was imposed at the cage level, it is unclear whether individual birds or cages were considered independent experimental units. This raises concerns regarding possible pseudoreplication and the validity of statistical inference. Additionally, the manuscript provides no direct evidence that the 10% increase in stocking density effectively induced a biologically meaningful stress response. No measurements of corticosterone, oxidative stress biomarkers, immune function, behavioral responses, mortality, or performance indicators were included to validate the stress model. Further concerns include the absence of information regarding sample size determination, randomization procedures, statistical power, and justification for the large number of comparisons performed. Although normality tests and ANOVA procedures are mentioned, the statistical models and interactions among factors are not described in sufficient detail. These limitations reduce reproducibility and weaken confidence in the conclusions drawn from the study.

Results and Discussion: The manuscript presents a large volume of data regarding amino acid composition across different ages, muscle types, sexes, and treatment groups. The breadth of the dataset represents one of the strengths of the study. Nevertheless, the presentation and interpretation of the results require considerable improvement. Much of the discussion simply restates numerical differences reported in the tables rather than providing meaningful biological interpretation. The extensive use of multiple statistical comparisons and complex superscript notation makes several tables difficult to interpret and reduces readability. The authors frequently attribute observed differences to stress adaptation or beneficial effects of the adaptogen complex; however, these interpretations remain largely speculative because no physiological measurements were collected to support the proposed mechanisms. For example, improvements in amino acid preservation are repeatedly linked to antioxidant activity and mitigation of oxidative stress, yet no indicators of oxidative status, antioxidant capacity, or endocrine responses were measured. Similarly, age was identified as the primary factor influencing amino acid composition, which is biologically plausible, but the physiological basis for these developmental changes is not sufficiently explored. Although the use of multifactorial ANOVA and eta-squared analysis is a positive aspect of the study, these results are not fully integrated into the biological discussion. The manuscript would benefit substantially from a more critical interpretation of the findings, greater emphasis on biological relevance rather than statistical significance alone, and stronger integration between the observed amino acid changes and the physiological processes underlying muscle development and stress adaptation.

Conclusions: The conclusions generally reflect the main findings of the study and correctly identify age as the principal factor influencing amino acid composition in broiler muscle tissue. The observation that supplementation with the adaptogen complex was associated with certain improvements in amino acid profiles under increased stocking density is supported by the reported data. However, several conclusions extend beyond the evidence generated by the study. In particular, statements suggesting that the adaptogen complex mitigated stress-induced effects should be interpreted with caution because stress itself was not directly quantified. Likewise, claims regarding preservation of meat nutritional quality are not fully substantiated by discussion of the biological relevance of the observed amino acid differences. The manuscript would benefit from a more explicit acknowledgment of its limitations, including the absence of physiological stress indicators, uncertainties regarding experimental replication, and the lack of mechanistic measurements. Future studies incorporating biochemical, endocrine, oxidative, and molecular markers would provide a more comprehensive understanding of how stocking density and adaptogen supplementation influence amino acid metabolism and meat quality in broiler chickens.

Comments on the Quality of English Language

The manuscript requires substantial English language revision before it can be considered for publication. Although the overall meaning is generally understandable, numerous grammatical errors, incorrect verb constructions, awkward sentence structures, and non-standard scientific expressions are present throughout the text. Several sentences appear to be direct translations and do not follow conventional English scientific writing style. Examples include incorrect phrases such as “It was been established”, “The adaptogen complex using”, and inconsistent use of articles, verb tenses, and terminology. In addition, the manuscript contains repetitive wording, excessively long sentences, formatting inconsistencies, and statistical annotations that reduce readability. The Results and Discussion sections would particularly benefit from language editing aimed at improving clarity, conciseness, and scientific precision. A thorough revision by a native English-speaking scientific editor or a professional language-editing service is strongly recommended.

Author Response

Dear Reviewer,

We are grateful for your thorough analysis of our work and for the comments you provided, which have improved our manuscript. We have carefully considered and incorporated all of your suggestions.

The research presented in this manuscript investigates the amino acid composition of breast and thigh muscles of Smena-9 broiler chickens reared under increased stocking density and supplemented with a complex of adaptogens consisting of dihydroquercetin, vitamin E, and vitamin C. The topic is relevant because intensive poultry production systems frequently expose birds to environmental stressors that may compromise growth performance, welfare, and meat quality. Furthermore, the nutritional quality of poultry meat, particularly its amino acid composition, represents an important aspect of consumer health and product value. The study has several strengths, including the evaluation of amino acid profiles in different muscle types, the consideration of age-related changes throughout ontogeny, and the investigation of a nutritional intervention intended to mitigate stress effects. However, several methodological, analytical, and interpretative limitations reduce the scientific impact of the manuscript. The most important concerns include uncertainty regarding the true experimental unit used in the statistical analyses, limited validation of the stocking-density stress model, excessive reliance on descriptive comparisons among amino acids, insufficient mechanistic explanations linking stress and amino acid metabolism, and a large number of statistical comparisons without discussion of potential inflation of Type I error. Additionally, the manuscript suffers from organizational issues, extensive and repetitive result descriptions, and numerous English language deficiencies that impair readability. Although the dataset generated is extensive and potentially valuable, substantial revisions aimed at improving methodological clarity, strengthening biological interpretation, enhancing statistical rigor, and improving scientific writing are necessary before the manuscript can be considered for publication.

Title: The title is descriptive and adequately reflects the principal variables investigated in the study, including amino acid composition, ontogenetic development, stocking density, and adaptogen supplementation. However, it is excessively long and somewhat difficult to read. In addition, the expression “correction with a complex of adaptogens” is unusual in scientific English and does not clearly communicate the nature of the intervention. The title emphasizes all experimental factors simultaneously, which reduces its focus and impact. A shorter and more precise title highlighting the effects of increased stocking density and adaptogen supplementation on amino acid composition in broiler meat would improve clarity and scientific appeal.

We agree with the reviewer's opinion and wanted to reflect all research topics to make it clear to the reader. We have now shortened the title to the following «Amino acid composition of chicken meat of the Smena 9 under increased stocking density and with adaptogens complex»

Abstract: The abstract provides a general overview of the objectives, experimental design, and principal findings of the study. The authors clearly describe the treatments and summarize the major observations regarding age-related changes and the effects of adaptogen supplementation. However, several shortcomings are evident. The biological rationale supporting the study is not sufficiently developed, and the knowledge gap being addressed is only briefly mentioned. The conclusions tend to be stronger than the evidence generated by the study. In particular, the authors suggest that the adaptogen complex compensates for stress-induced alterations in meat quality, yet no direct physiological, endocrine, oxidative, or welfare-related indicators were measured to confirm the occurrence of stress or the mechanisms through which the additives exerted their effects. Furthermore, numerous numerical findings are reported without prioritizing those that are biologically most meaningful. As a result, the abstract would benefit from a more critical interpretation of the findings and a clearer acknowledgment of the study’s limitations.

We thank the reviewer for this critical observation. The reviewer correctly notes that our primary study focused on amino acid composition and did not include direct measurements of physiological, endocrine, oxidative, or welfare-related parameters. We fully acknowledge this limitation. However, we would like to clarify that the same experimental model and bird cohorts were used in our parallel studies, which specifically addressed these aspects. We believe these complementary datasets provide a robust foundation for interpreting the amino acid changes observed in the present work.

Thus, the early administration of the adaptogens complex appears to mitigate for high stocking density negative effects and contributes to preserving the nutritional value of broiler meat, although the underlying mechanisms remain hypothetical and require further.

Introduction: The introduction provides substantial background information regarding poultry meat composition, amino acid functionality, stress physiology, and antioxidant supplementation. Nevertheless, the section is predominantly descriptive and lacks sufficient analytical depth. The physiological mechanisms through which stocking density may influence muscle growth, protein deposition, amino acid metabolism, and meat quality are discussed only superficially. Although oxidative stress is repeatedly mentioned, the manuscript does not adequately explain how oxidative damage could specifically alter amino acid composition within muscle tissue. The literature review includes studies involving amino acid profiles and antioxidant supplementation, but there is limited integration of previous findings and little discussion regarding conflicting results reported in the literature. The authors identify a lack of information regarding amino acid composition under different stocking densities; however, the scientific hypothesis remains broad and insufficiently mechanistic. Stronger synthesis of previous studies and a more explicit biological framework would substantially improve the introduction.

We thank the reviewer for this detailed and constructive comment. We fully agree that the initial version of the Introduction was primarily descriptive and lacked sufficient analytical depth regarding the mechanistic links between stress caused by weight loss, amino acid metabolism in muscles, and the potential role of antioxidant adaptogens. In response to this comment, we have significantly revised and structured the Introduction.

We have added the following

High stocking density, like other types of stress, leads to activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased corticosterone secretion [16,17]. This hormone can exert a dual effect on protein metabolism: it activates the ubiquitin-proteasome pathway (upregulating MuRF-1 and Atrogin-1/MAFbx), stimulating protein degradation, and suppresses the IGF‑1/Akt/mTOR signaling pathway, inhibiting protein synthesis [17,18]. Furthermore, increased glucocorticoid production stimulates gluconeogenesis in the liver, utilizing amino acids—particularly alanine and glutamine—as substrates, thereby reducing their availability for deposition in muscle tissue [19]. In addition, high stocking density may restrict bird mobility, leading to mitochondrial dysfunction in skeletal muscles, impaired oxidative phosphorylation, and altered energy metabolism, which in turn affects amino acid utilization and protein turnover [20]

It is known that oxidative stress can affect the amino acid composition in the following ways. First, the side chains of amino acids are directly oxidized and undergo oxidative modification. Oxidized amino acids cannot be reused for protein synthesis. Secondly, the process of protein carbonylation makes them more susceptible to proteolytic degradation. Thirdly, lipid peroxidation products react with nucleophilic amino groups, reducing their bioavailability for protein synthesis. Fourth, oxidative stress depletes glutathione (GSH), the main intracellular antioxidant, increasing the metabolic demand for its precursor amino acids— cysteine, glutamate, and glycine [25]. Fifth, mitochondrial dysfunction induced by oxidative stress disrupts the tricarboxylic acid (CTC) cycle, affecting the synthesis of interchangeable amino acids formed from intermediates [31]. Thus, oxidative stress creates conditions for changing the amino acid composition of muscle tissue through direct oxidation of specific residues, increased protein turnover, and competitive redistribution of amino acids for antioxidant protection, reducing their availability to structural proteins.
We hypothesized increased stocking density may alter the amino acid composition of muscle tissue in a muscle-type- and age-dependent manner. Dietary supplementation with the DHQEC complex (dihydroquercetin + vitamins E and C) is expected to mitigate these effects. Furthermore, we propose the efficacy of the adaptogens complex depends on the timing of its administration, with earlier supplementation (from day 1) providing greater protection than later supplementation (from day 21).

Materials and Methods: The experimental design combines nutritional intervention and environmental challenge in an attempt to evaluate their effects on amino acid composition in broiler meat. The description of the amino acid analytical procedures is detailed and appears technically appropriate, particularly the use of ion-exchange chromatography with post-column derivatization. However, several methodological aspects require clarification. The most important issue concerns the experimental unit used in the statistical analyses. Because stocking density was imposed at the cage level, it is unclear whether individual birds or cages were considered independent experimental units. This raises concerns regarding possible pseudoreplication and the validity of statistical inference. Additionally, the manuscript provides no direct evidence that the 10% increase in stocking density effectively induced a biologically meaningful stress response. No measurements of corticosterone, oxidative stress biomarkers, immune function, behavioral responses, mortality, or performance indicators were included to validate the stress model. Further concerns include the absence of information regarding sample size determination, randomization procedures, statistical power, and justification for the large number of comparisons performed. Although normality tests and ANOVA procedures are mentioned, the statistical models and interactions among factors are not described in sufficient detail. These limitations reduce reproducibility and weaken confidence in the conclusions drawn from the study.

We thank the reviewer for a thorough analysis of the work. We agree with the comments made and have made some adjustments to this section.:

Added information about the bird's landing density
The ANOVA method has specified.

Also in the introduction, we discussed the issue of studying the increase in planting density on the state of antioxidant, hormonal status, biochemical and molecular genetic markers, providing references and the results of the studies obtained. Therefore, it is not advisable to repeat them in this publication. As for the sample size, it is specified in the section.

The planting density changed as the bird grew. On day 21, the bird planting density in the S(-)CON group was 38 heads per 1 m², in the other groups it was increased by 10% and amounted to 42 heads per 1 m². Each week, the planting density changed and amounted to 32 and 35 heads per 1 m² in 28-35 days, 21 and 23 heads per 1 m² in 35-42 days, and 17 and 19 heads per 1 m² in 43 days and until the end of fattening, respectively. The area of one cage was 0.6 m²(0.99 cm long and 0.61 cm wide). In the initial period, with a landing density of 38 birds per 1 m² (S(-)CON group), 23 birds were kept in one cage, and the remaining 17 birds (40-23=17) were kept in another cage with an area of 0.44 m² (0.99 cm long and 0.44 cm wide, achieved by shifting a temporary plywood partition). In a group with a planting density of 42 heads (S(+)CON), 25 birds were kept in 1 cage with an area of 0.6 m², and the remaining 15 (40-25=15) were kept in a cage with an area of 0.36 m² (length 0.99 cm, width 0.36 cm, which was achieved by shifting a temporary plywood partition). The same principle was used for planting birds in other periods.

Results and Discussion: The manuscript presents a large volume of data regarding amino acid composition across different ages, muscle types, sexes, and treatment groups. The breadth of the dataset represents one of the strengths of the study. Nevertheless, the presentation and interpretation of the results require considerable improvement. Much of the discussion simply restates numerical differences reported in the tables rather than providing meaningful biological interpretation. The extensive use of multiple statistical comparisons and complex superscript notation makes several tables difficult to interpret and reduces readability. The authors frequently attribute observed differences to stress adaptation or beneficial effects of the adaptogen complex; however, these interpretations remain largely speculative because no physiological measurements were collected to support the proposed mechanisms. For example, improvements in amino acid preservation are repeatedly linked to antioxidant activity and mitigation of oxidative stress, yet no indicators of oxidative status, antioxidant capacity, or endocrine responses were measured. Similarly, age was identified as the primary factor influencing amino acid composition, which is biologically plausible, but the physiological basis for these developmental changes is not sufficiently explored. Although the use of multifactorial ANOVA and eta-squared analysis is a positive aspect of the study, these results are not fully integrated into the biological discussion. The manuscript would benefit substantially from a more critical interpretation of the findings, greater emphasis on biological relevance rather than statistical significance alone, and stronger integration between the observed amino acid changes and the physiological processes underlying muscle development and stress adaptation.

Thanks to the reviewer for these comments. The effect of stocking density on blood biochemical parameters, antioxidant status, and hormonal status was reflected in our recent publication [22]. In this study, changes in the level of cortisol and the activity of antioxidant enzymes in the blood had the character of trends (p > 0.05), which does not allow us to assert the presence of systemic stress with complete statistical certainty. However, the same parallel study revealed statistically significant changes in key biochemical parameters consistent with stress-induced metabolic alterations. By day 34, birds in the high-stocking-density group showed a significant increase in glucose (+4.1%, p = 0.005) and total bilirubin (+31.7%, p = 0.02). By day 52, we observed a significant increase in total protein (+4.5%, p = 0.001) and glucose (+4.4%, p = 0.006), along with a decrease in albumin (-1.5%, p < 0.001) and AST activity (-26.4%, p = 0.007), as well as an increase in triglycerides (+5.8%, p = 0.005) [23]. These changes—hyperglycemia, hyperbilirubinemia, dysproteinemia, and elevated triglycerides—are well-documented indicators of systemic stress, reflecting glucocorticoid-mediated gluconeogenesis, oxidative damage, inflammatory response, and lipid mobilization in poultry subjected to adverse environmental conditions. Furthermore, the results of our other work [24] demonstrated statistically significant suppression of the expression of immunity and antioxidant protection genes Nrf2, GSH-Gpx, and HO-1 in the intestinal and liver tissues of broiler chickens under high stocking density. Decreased expression of these key antioxidant enzymes is a generally recognized indicator of oxidative stress induced by chronic exposure to adverse factors. Oxidative stress is considered one of the main mechanisms of the pathogenesis of stress reactions in birds. The positive correlation we found (r = 0.983, p < 0.05) between AvBD9 expression in the caecum and average daily gain confirms that the suppression of protective mechanisms we observed is associated with decreased productivity, a classic integral indicator of chronic stress in animals and poultry.

We have made a number of additions to the discussion of the results.

Conclusions: The conclusions generally reflect the main findings of the study and correctly identify age as the principal factor influencing amino acid composition in broiler muscle tissue. The observation that supplementation with the adaptogen complex was associated with certain improvements in amino acid profiles under increased stocking density is supported by the reported data. However, several conclusions extend beyond the evidence generated by the study. In particular, statements suggesting that the adaptogen complex mitigated stress-induced effects should be interpreted with caution because stress itself was not directly quantified. Likewise, claims regarding preservation of meat nutritional quality are not fully substantiated by discussion of the biological relevance of the observed amino acid differences. The manuscript would benefit from a more explicit acknowledgment of its limitations, including the absence of physiological stress indicators, uncertainties regarding experimental replication, and the lack of mechanistic measurements. Future studies incorporating biochemical, endocrine, oxidative, and molecular markers would provide a more comprehensive understanding of how stocking density and adaptogen supplementation influence amino acid metabolism and meat quality in broiler chickens.

We agree with the reviewer's comment and have added the following information to the conclusion:

We acknowledge that the mechanistic interpretations proposed in this study are based on established physiological pathways and are supported by our parallel biochemical and molecular data [22,24]; however, direct measurements of systemic stress hormones, circulating reactive oxygen species, or muscle protein degradation markers were not performed in the present work. Therefore, the specific mechanisms linking the observed amino acid changes to systemic stress responses remain hypothetical. Further targeted studies are needed to directly quantify glucocorticoid receptor signaling, oxidative stress markers in muscle tissue, and protein turnover rates under crowding stress

Comments on the Quality of English Language

Many thanks to the reviewers for the low quality of the English language. The material of the article was revised and the quality of the language has been improved.

Reviewer 4 Report

Comments and Suggestions for Authors

Overall Evaluation

This manuscript focuses on the effects of high stocking density and a compound DHQEC feed additive supplemented at different administration ages on muscle amino acid profiles of Smena9 broilers. The research targets a practical problem in intensive poultry production, features well-organized experimental grouping and standardized amino acid detection, and delivers actionable feeding suggestions for commercial broiler farming. Overall, the work is scientifically valuable and recommended for revision prior to formal acceptance.

Vague stress modeling: "Simulated environmental conditions" is never operationally defined. The manuscript states HSD was increased by 10% from day 21, but absolute bird density (birds/m² or cm²/bird) is missing. Table 3 provides general guidelines, but not the actual density used.

Information about cage replicates is ambiguous in methodology. It is unclear whether the 10 sampled birds per group serve as individual replicates or cage replicates; the exact number of breeding cages per treatment needs complementary description.

Statistical Reporting & Transparency

Missing key information: No power analysis is reported to justify n=10 per group per time point. The number of cages, birds per cage, and whether individual bird or cage was the experimental unit are not stated.

Multiple comparison issues: Tukey’s post-hoc tests were used, but the letter-based significance notation (a, b, c, d) in Tables 5–7 is confusing and appears to have typographical errors (e.g., superscripts attached to numbers incorrectly).

Results Section – Overinterpretation & Redundancy

Redundant text: The Results section repeats almost all numerical values from the tables instead of summarizing key trends.

Claim without support: The statement “The improvement in the amino acid profile … may be due to the body's adaptation to stress conditions as well as the positive effect of the adaptogen complex supplement” (lines 262–266) is speculative and placed in Results instead of Discussion. This belongs in the Discussion.

Discussion Section – Overclaiming & Weak Citations

Unsubstantiated mechanisms: The authors repeatedly invoke “corticosterone” and “oxidative stress” as explanations, but no measurements of corticosterone, ROS, antioxidant enzymes, or protein degradation markers were made. These remain hypotheses, not conclusions.

Economic benefit analysis of the additive is not performed. Cost of DHQEC input and corresponding premium of high-quality broiler meat are not calculated, weakening the economic persuasion of application recommendations.

Language, Formatting, & Clarity

Poor English grammar & syntax: Numerous sentences are awkward or incorrect, e.g.:

“It was been established” (line 20) → “It has been established”.

References are numbered but text often cites names + year (e.g., “Bychaev A.G. (2019)” . Ensure all in-text citations appear in reference list and follow journal format.

Author Response

Dear Reviewer,

We are grateful for your thorough analysis of our work and for the comments you provided, which have improved our manuscript. We have carefully considered and incorporated all of your suggestions.

We agree with the reviewer's opinion that it is necessary to specify the number of individuals per 1 square meter. The planting density changed as the bird grew. On day 21, the bird planting density in the S(-)CON group was 38 heads per 1 m², in the other groups it was increased by 10% and amounted to 42 heads per 1 m². Each week, the planting density changed and amounted to 32 and 35 heads per 1 m² in 28-35 days, 21 and 23 heads per 1 m² in 35-42 days, and 17 and 19 heads per 1 m² in 43 days and until the end of fattening, respectively. The area of one cage was 0.6 m²(0.99 cm long and 0.61 cm wide). In the initial period, with a landing density of 38 birds per 1 m² (S(-)CON group), 23 birds were kept in one cage, and the remaining 17 birds (40-23=17) were kept in another cage with an area of 0.44 m² (0.99 cm long and 0.44 cm wide, achieved by shifting a temporary plywood partition). In a group with a planting density of 42 heads (S(+)CON), 25 birds were kept in 1 cage with an area of 0.6 m², and the remaining 15 (40-25=15) were kept in a cage with an area of 0.36 m² (length 0.99 cm, width 0.36 cm, which was achieved by shifting a temporary plywood partition). The same principle was used for planting birds in other periods.

Statistical Reporting & Transparency

Thanks to the reviewer for the question. The answer to this remark is presented above.

We have checked all the data and fixed typographical errors.

Results Section – Overinterpretation & Redundancy. Redundant text: The Results section repeats almost all numerical values from the tables instead of summarizing key trends.

Indeed, all the data is presented in tables. We describe the data for greater clarity and understanding for readers. Generalizations and key conclusions are given in the Discussion of the results section.

This remark was eliminated.

This text was been moved to the discussion.

The improvement in the amino acid profile, resulting from both an increase in the total amino acid content and individual functional amino acids such as methionine, phenylalanine, histidine and arginine, may be attributed to the birds' adaptation to stress conditions and the positive effect of the adaptogen complex supplementation.

Discussion Section – Overclaiming & Weak Citations

We completely agree with the reviewer. This is a critical limitation of our study. We did not measure corticosterone, ROS, or antioxidant enzyme activities in the present work.

The effect of stocking density on blood biochemical parameters, antioxidant status, and hormonal status was reflected in our recent publication (Bogolyubova N.V., Nekrasov R.V., Lakhonin P.D., Kolesnik N.S., V'yuchnaya P.S., Zelenchenkova A.A., Nikanova D.A., Bogolyubova Yu.A. Biological and productive effect of a complex of adaptogens on broiler chickens (Gallus gallus L.) of Smena 9 cross under increased stocking density. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2026, 61(2), 304–319. https://doi.org/10.15389/agrobiology.2026.2.304rus). In this study, changes in the level of cortisol and the activity of antioxidant enzymes in the blood had the character of trends (p > 0.05), which does not allow us to assert the presence of systemic stress with complete statistical certainty. We acknowledge that these observations warrant further investigation on a larger poultry population. We admit that the use of the term "stress" as an established fact in the current manuscript was unnecessarily categorical, since neither in this nor in our previous work did we obtain strict statistically significant differences in classical markers of systemic stress (cortisol, lipid peroxidation products).

However, the same parallel study revealed statistically significant changes in key biochemical parameters consistent with stress-induced metabolic alterations. By day 34, birds in the high-stocking-density group showed a significant increase in glucose (+4.1%, p = 0.005) and total bilirubin (+31.7%, p = 0.02). By day 52, we observed a significant increase in total protein (+4.5%, p = 0.001) and glucose (+4.4%, p = 0.006), along with a decrease in albumin (-1.5%, p < 0.001) and AST activity (-26.4%, p = 0.007), as well as an increase in triglycerides (+5.8%, p = 0.005)( The influence of simulated stress on the biochemical parameters of broiler chickens / P. D. Lakhonin, N. V. Bogolyubova, R. V. Nekrasov [et al.] // Veterinary medicine and feeding. – 2025. – No. 6. – pp. 62-67. – DOI 10.30917/TT-VK-1814-9588-2025-6-14.). These changes—hyperglycemia, hyperbilirubinemia, dysproteinemia, and elevated triglycerides—are well-documented indicators of systemic stress, reflecting glucocorticoid-mediated gluconeogenesis, oxidative damage, inflammatory response, and lipid mobilization in poultry subjected to adverse environmental conditions.

Furthermore, the results of our other work (Expression of antioxidant protection and immunity genes in the tissues of broiler chickens under the influence of a complex of adaptogens / P. S. Vyuchnaya, P. D. Lakhonin, N. V. Bardukov, N. V. Bogolyubova // International Bulletin of Veterinary Medicine. – 2025. – No. 2. – pp. 176-189. – DOI 10.52419/issn2072-2419.2025.2.1766.) demonstrated statistically significant suppression of the expression of immunity and antioxidant protection genes Nrf2, GSH-Gpx, and HO-1 in the intestinal and liver tissues of broiler chickens under high stocking density. Decreased expression of these key antioxidant enzymes is a generally recognized indicator of oxidative stress induced by chronic exposure to adverse factors. Oxidative stress is considered one of the main mechanisms of the pathogenesis of stress reactions in birds. The positive correlation we found (r = 0.983, p < 0.05) between AvBD9 expression in the caecum and average daily gain confirms that the suppression of protective mechanisms we observed is associated with decreased productivity, a classic integral indicator of chronic stress in animals and poultry.

Our experimental data directly support tissue-level transcriptional changes, performance parameters, and systemic metabolic shifts consistent with stress response. Nevertheless, we fully acknowledge that direct measurements of corticosterone, circulating ROS, and muscle proteolysis markers were not performed in the present work, and therefore the specific mechanisms linking these systemic changes to the amino acid profile remain speculative. Further studies with direct quantification of glucocorticoid signaling, oxidative stress markers, and protein turnover rates are needed to establish causal relationships.

To address the reviewer's concern, we have made the following revisions to the text:

In the Introduction: While the present study focuses on amino acid composition, our parallel investigations using the same experimental model have revealed systemic biochemical alterations (hyperglycemia, increased bilirubin and triglycerides, decreased albumin) and tissue-level molecular changes (suppressed expression of Nrf2, GSH-Gpx, and HO-1) consistent with stress-related metabolic shifts [22,23,24]. These findings will be considered in the interpretation of the observed amino acid profile dynamics.

In the Discussion

1.Broiler chickens are considered more susceptible to adverse environmental conditions, likely due to their higher metabolic rate associated with rapid growth [24]. A comparison of the S(-)CON and S(+)CON groups indicated that the housing conditions with increased stocking density, which are generally considered stressful in the literature [14, 25], affected the amino acid composition of the breast and thigh of broiler chickens in our study, manifesting itself at 34 days of age (2 weeks after the start of exposure).

It should be noted, however, that our parallel study did not reveal statistically significant changes in serum corticosterone levels in the same birds [22]; therefore, the involvement of systemic glucocorticoid signaling in the observed amino acid changes remains speculative. More convincingly, our independent molecular data demonstrated significant downregulation of antioxidant defense genes (Nrf2, GSH-Gpx, HO-1) in intestinal and liver tissues under high stocking density [24], suggesting local oxidative stress at the tissue level, which may contribute to altered amino acid metabolism.
This may lead to a reduced requirement for methionine — which plays a key role in the antioxidant defense system — for these purposes, potentially contributing to its increased content in muscle tissue. However, direct evidence linking these changes to decreased oxidative stress in muscle tissue was not obtained in the present study and requires further investigation.

4.We acknowledge that the mechanistic interpretations proposed in this study are based on established physiological pathways and are supported by our parallel biochemical and molecular data [22-24]; however, direct measurements of systemic stress hormones, circulating reactive oxygen species, or muscle protein degradation markers were not performed in the present work. Therefore, the specific mechanisms linking the observed amino acid changes to systemic stress responses remain hypothetical. Further targeted studies are needed to directly quantify glucocorticoid receptor signaling, oxidative stress markers in muscle tissue, and protein turnover rates under crowding stress

We completely agree with the reviewer's comment. The objective of our research was not to calculate the economic efficiency of using this complex. The main purpose of the development of this complex was to strengthen the immune, biochemical status and improve product quality. It is expected that there will be an economic effect, since previous studies have found an increase in the gross and average daily increase in live weight of chickens after using the complex to 5.6%. Preliminary calculations show a positive balance, but they require more calculations, which will be done in further studies. The complex of adaptogens contributed to a decrease in oxidative stress in the body by increasing the activity of antioxidant enzymes, reducing the levels of cortisol and lipid peroxidation products in the blood.

Language, Formatting, & Clarity.Poor English grammar & syntax: Numerous sentences are awkward or incorrect, e.g.: “It was been established” (line 20) → “It has been established”. References are numbered but text often cites names + year (e.g., “Bychaev A.G. (2019)” . Ensure all in-text citations appear in reference list and follow journal format.

Many thanks to the reviewers for the low quality of the English language. The material of the article was revised and the quality of the language has been improved. All references are checked and presented in the list of references

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors
I greatly admire your efforts to significantly improve the manuscript compared to the previous version. The manuscript has improved; however, several issues still require clarification.

Title

I suggest changing the following change in title for better word flow “Amino acid composition of Smena 9 chicken meat with adaptogens complex under increased stocking density”

As per the title, the study will focus on the investigation of the effect of stocking density and the adaptogens complex. However, the manuscript also extensively evaluates the effects of age and sex. Consequently, the title does not fully reflect the scope of the study. The authors should either revise the title to include all major experimental factors or reconsider the study focus and place greater emphasis on the primary research question.

Abstracts

The abstract contains excessive methodological detail, whereas the key findings are not sufficiently highlighted. It is suggested to increase the proportion of results, particularly changes in amino acids, under different treatments. Specify the treatments that improved particular amino acids.

Materials and Methods

The manuscript states that 30 birds were allocated to each treatment group. However, the number of experimental replicates is not clearly described. Please specify the number of cages/replicates per treatment and clearly identify the experimental unit used in the statistical analysis. Considering that males and females were analyzed separately and ten birds per treatment were sampled at each age, the effective sample size appears to be approximately five birds per sex within each treatment-age combination. The authors should justify whether this sample size provides sufficient statistical power to detect biologically meaningful differences.

The cage area is reported as 0.60 m²; therefore, it would be more informative to report the actual number of birds housed per cage rather than presenting stocking density only on a birds/m² basis. This would allow readers to better understand the experimental conditions.

Table 3 requires clarification. Does it show stocking density standard for Smena 9 chicken or conventional broiler standards? The source of these recommendations should be clearly stated and referenced.

Rather than stating that temperature and humidity were maintained according to standard recommendations, it would be preferable to report the actual temperature and relative humidity ranges recorded during the experiment.

I suggest the authors should revisit their research question and hypothesis. If they are investigating the effect of stocking density and adaptogens, they shall focus on the comparison of these two parameters and, if necessary, add the comparison of male and female. They shall avoid comparison of age as a source of variation, as answering so many problems in one study distracts the focus from the main problem. It is suggested to just compare the main treatments (adaptogens with high stocking densities) at various ages.

Results

The notation system (a, aa, aaa, b, bb, etc.) remains difficult to interpret. The tables would be clearer if a simpler and more conventional superscript system were used. Amino acid comparisons should be presented consistently within each row, with superscripts clearly indicating significant differences among treatments.

Line 695: Use “Stocking density” instead of “Housing conditions”. As you just focused on one aspect of housing, not all.

Avoid using references in the conclusion section.

The titles of the tables require substantial improvement. Each table title should be self-explanatory and provide sufficient information for readers to understand the content without referring extensively to the main text.

Discussion

Line 458: The authors mention that the corticosterone levels in the parallel study were not affected by the stocking densities, which again raises the concern about the level of stress induced by 10% increase in stocking density and subsequent use of adaptogens.

The Discussion would benefit from greater emphasis on the practical significance of the observed amino acid changes. Although several differences were statistically significant, the authors should clarify whether these differences are large enough to be nutritionally or commercially meaningful.

Author Response

Dear Reviewer,

We are grateful for your thorough analysis of our work and for the comments you provided, which have improved our manuscript. We have carefully considered and incorporated all of your suggestions.

Changes made to the manuscript are highlighted in green.

1.Title. I suggest changing the following change in title for better word flow “Amino acid composition of Smena 9 chicken meat with adaptogens complex under increased stocking density”

We agree with the reviewer's comment and aimed to reflect all aspects of the study clearly for the reader. We have revised the title as follows: "Amino acid composition of meat from Smena 9 broiler chickens during ontogeny under a high stocking density and dietary adaptogens complex." We would also be very grateful if the reviewer, should they still find the title unsatisfactory, could suggest how it might be further improved.

2.Abstracts

The Results section of the abstract should be presented more specifically. For example, housing conditions affected amino acid composition (line 46) is a very vague statement. The study specifically evaluated stocking density rather than overall housing conditions. Therefore, the terminology should accurately reflect the experimental treatments investigated. The abstract contains excessive methodological detail, whereas the key findings are not sufficiently highlighted. It is suggested to increase the proportion of results, particularly changes in amino acids, under different treatments. Specify the treatments that improved particular amino acids.

We thank the reviewer for this precise observation. We fully agree that the term "housing conditions" is too broad and does not accurately reflect the specific experimental factor that was manipulated in our study. The only housing parameter that differed between groups was stocking density. Accordingly, we have revised the indicated sentence in the Abstract (line 47), replacing the vague term "housing conditions" with the specific term "stocking density." We believe that this correction makes the Abstract more precise and accurately reflects the experimental design. We are grateful to the reviewer for this valuable clarification.

We sincerely thank the reviewer for this valuable and constructive suggestion. We fully agree that the original Abstract was disproportionately focused on methodological details, while the quantitative amino acid results were underemphasized. To address this comment, we have revised the Abstract by adding three concise, data-driven sentences that provide specific numerical results and clearly identify the treatments responsible for the amino acid improvements, while preserving the overall structure of the original text. Specifically, these additions include: a quantitative comparison between the breast and thigh muscles, specification of the treatment effects in the S(+)DHQEC_1 group, and the stabilization of flavor-related amino acids (SFAA) in the S(+)DHQEC_1 group.

Changes made to the manuscript:

We have revised the Abstract on lines 49-50 and 53-57. The revised version now reads as follows (new additions are highlighted in green).

3.Materials and Methods

We thank the reviewer for these valuable comments, which have helped us improve the clarity of our experimental design and statistical approach.

Experimental replicates and sample size. We agree that the description of replicates was insufficient. In our experimental design, birds were housed in cages with a floor area of 0.60 m². Stocking density was adjusted weekly by moving temporary partitions to maintain the target density as the birds grew. For each treatment, 30 birds were used for sampling and analysis (10 birds at each of the three slaughter ages: days 24, 34, and 52). At each sampling point, we randomly selected 5 males and 5 females per treatment to ensure balanced sex representation. The experimental unit for all statistical analyses was the individual bird, as birds were randomly sampled from the population and all measurements were performed on individual animals. This information has been added to the Materials and Methods section (lines 194–199).

Stocking density – birds per cage.

The reviewer is correct that reporting only birds/m² is insufficient. We have therefore added the following information to the Housing subsection: "Birds were housed in cages with a floor area of 0.60 m² (length 0.99 m × width 0.61 m). Stocking density was adjusted weekly by moving temporary plywood partitions to maintain the target density as the birds grew. The number of birds per cage was as follows: during days 0–21, 23 birds per cage in the S(-)CON group (38 birds/m²) and 25 birds per cage in the S(+)CON group (42 birds/m²); during days 22–28, 19 birds per cage (32 birds/m²) and 21 birds per cage (35 birds/m²), respectively; during days 29–35, 19 and 21 birds per cage; during days 36–42, 13 and 14 birds per cage (21 and 23 birds/m², respectively); and from day 43 to 52, 10 and 11 birds per cage (17 and 19 birds/m², respectively). All cages within the same treatment group were maintained under identical environmental conditions (temperature, humidity, lighting, and ventilation) throughout the experiment."

This information has been added to the Housing subsection (lines 200–208) to ensure full transparency of the experimental conditions.

Sex distribution and balanced sampling. At each of the three slaughter ages (days 24, 34, and 52), 10 birds per treatment were randomly selected for necropsy and sample collection. From day 24 onward, sex was determined visually prior to slaughter based on comb size and coloration, which are reliably distinguishable in broilers by this age. This allowed us to deliberately select 5 males and 5 females per treatment at each sampling point, ensuring a balanced subsample for sex-specific analyses.

This information has been added to the Sampling subsection (lines 212–216).

Statistical analysis. Prior to ANOVA, normality of the data distribution was verified using the Shapiro–Wilk test, which is recommended for small sample sizes (n = 5). Homogeneity of variances was assessed using Levene's test. Since all data met the assumptions of normality and homoscedasticity, one-way ANOVA was applied, followed by Tukey–Kramer post-hoc tests for multiple comparisons. The Tukey–Kramer procedure was used to control the family-wise error rate (type I error) associated with multiple pairwise comparisons. To justify the adequacy of our sample size, we performed a post-hoc power analysis based on the primary response variables. With n = 5 birds per sex × treatment × age combination, and using the observed means and pooled standard deviations, the achieved statistical power exceeded 80% at α = 0.05 for detecting the observed effect sizes.

This information has been added to the Statistical Analysis subsection (lines 249–259).

We believe that these revisions adequately address the reviewer's concerns and substantially improve the transparency and reproducibility of our study.

We thank the reviewer for this important and constructive suggestion. This comment is entirely justified. The values presented are indeed the standard stocking density guidelines for poultry. We have now indicated this in the table title and added a reference to the source.

We thank the reviewer for this important and constructive suggestion.

The temperature and humidity values corresponded to the standards recommended for broilers. During week 1, the average temperature was 32.5°C and the relative humidity was 55%; during weeks 2–3, the corresponding values were 22°C and 65%; during weeks 4–6, 21.9°C and 65%; and during week 7, 19°C and 65%.

We sincerely thank the reviewer for this thoughtful comment. We fully agree that the primary focus of our study is the effect of a high stocking density and adaptogen supplementation on the amino acid composition of broiler meat. This is indeed the central research question, and we have carefully designed the experiment and structured the manuscript to ensure that the treatment effects are clearly presented and discussed. At the same time, we respectfully believe that age cannot be excluded from the analytical framework, as it is a well-established factor influencing meat quality and amino acid composition. Age is not an independent object of study in our work, but rather a biological variable that we included to ensure the scientific rigor and practical relevance of our findings. Broiler muscle development and amino acid metabolism are inherently dynamic processes, and ignoring age-related variation would have limited our ability to interpret the treatment effects accurately. By assessing the amino acid composition at three developmental time points (24, 34, and 52 days), we were able to determine whether the effects of stocking density and adaptogen supplementation are consistent throughout the rearing period or manifest only at specific stages. This does not distract from our main question — it strengthens the validity of our conclusions by placing the treatment effects in their proper biological context. We also wish to emphasize that our study was not designed to compare age as a factor of primary interest, nor do we provide specific recommendations on slaughter age. Rather, we present age as a contextual factor that helps to better understand the dynamics of the treatment effects. The practical implication of including age is that it allows us to answer the question: "At what stage of development are the effects of adaptogens most pronounced, and when is the meat composition most favorable?" This is valuable information for poultry producers who seek to optimize their management strategies, but we do not prescribe a specific slaughter age based on our data alone.

Regarding sex-related comparisons, we included them only where statistically significant differences were observed (the breast muscle at 24 days), and our conclusion clearly states that sex effects are minimal and do not require separate management consideration. This is consistent with the reviewer's suggestion to limit sex comparisons to where they are relevant. To ensure that the manuscript accurately reflects this logic, we have revised the Discussion.

In this study, we evaluated the effects of stocking density and adaptogen supplementation at three developmental stages to capture the temporal dynamics of these effects and to ensure that our findings are placed in their proper biological context.

We are grateful to the reviewer for prompting us to articulate this rationale more explicitly, and we hope that the revised manuscript now clearly conveys that our study is first and foremost about the effects of stocking density and adaptogens, with age serving as a necessary temporal framework for their evaluation.

4.Results

We thank the reviewer for this important and constructive suggestion.

The data were statistically analyzed using Microsoft Office Excel 2003 and STATISTICA 10 (Statistica 13RU, StatSoft, Inc., USA) with descriptive statistics, analysis of variance, and factor analysis. Since the ANOVA revealed significant differences, Tukey's post-hoc test was applied to identify exactly where these differences occurred. This test controls the family-wise error rate (type I error) in multiple comparisons.

To present the results of pairwise comparisons clearly in the text and tables, a letter-based notation system is used. Letters indicate a statistically significant difference between a given group and the control or other groups. Each letter (a, b, c, d) is assigned to a specific group for comparison purposes. For example, the letter 'a' always refers to comparisons with Group 1 (control), the letter 'b' to comparisons with Group 2, and so on. The number of letters indicates the level of statistical significance of the observed difference.

Significance levels:

Designation | Significance level (p-value) | Degree of significance

a / b / c / d | p < 0.05 | Significant difference (standard level)

aa / bb / cc / dd | p < 0.01 | Highly significant difference

aaa / bbb / ccc / ddd | p < 0.001 | Very highly significant difference

The following explanatory notes are provided beneath each table:

Significant by Tukey's test with Group 1 (control): a – p < 0.05, aa – p < 0.01, aaa – p < 0.001

Significant by Tukey's test with Group 2: b – p < 0.05, bb – p < 0.01, bbb – p < 0.001

Significant by Tukey's test with Group 3: c – p < 0.05, cc – p < 0.01, ccc – p < 0.001

Significant by Tukey's test with Group 4: d – p < 0.05, dd – p < 0.01, ddd – p < 0.001

We believe that this notation system provides the most complete representation of the statistical significance of our results without any loss of information regarding the level of significance.

Line 695: Use “Stocking density” instead of “Housing conditions”. As you just focused on one aspect of housing, not all.

We thank the reviewer for this important and constructive suggestion.

We agree with the reviewer and have revised the text as follows (line 773):

"The results of this study demonstrate that the amino acid composition of the breast and thigh muscles of Smena 9 broiler chickens changed significantly depending on the slaughter age, stocking density, and the use of the adaptogen complex."

Avoid using references in the conclusion section.

We thank the reviewer for this important and constructive suggestion. This has been addressed in the Conclusions section.

We thank the reviewer for this important and constructive suggestion. The comment has been addressed. A correction has been made to Table 10.

5.Discussion

We thank the reviewer for this important and constructive suggestion.

Indeed, corticosterone levels were not addressed in the parallel study. While the present study focuses on amino acid composition, our parallel investigations using the same experimental model have revealed systemic biochemical alterations (hyperglycemia, increased bilirubin and triglycerides, decreased albumin) and tissue-level molecular changes (suppressed expression of Nrf2, GSH-Gpx, and HO-1) consistent with stress-related metabolic shifts [22–24].

We thank the reviewer for this valuable suggestion. In response, we have added a comprehensive new paragraph at the end of the Discussion section (lines 727-769) that explicitly evaluates the nutritional and commercial significance of our findings. This paragraph focuses solely on statistically significant changes and interprets them in the context of protein quality and the role of methionine as the first limiting amino acid, feed formulation economics (reduced need for synthetic methionine supplementation), and carcass value and sensory quality. We believe that this addition substantially strengthens the translational value of our study and provides clear, evidence-based recommendations for poultry producers and nutritionists.

Reviewer 3 Report

Comments and Suggestions for Authors

The quality of the manuscript has improved considerably. The text is much clearer and more comprehensive. In addition, the authors have adequately addressed or justified all of the suggested revisions. Therefore, I consider the manuscript suitable for publication.

Author Response

Dear Reviewer, we thank you for your attention to our manuscript and for your valuable and meaningful comments, which have undoubtedly improved the quality of our work. We wish you health and continued creative success.