Different Concentrations of Copaiba Oil (Copaifera spp.) in Ruminal Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work generates information about an ingredient that is beginning to be used in animal feed.

OBSERVATIONS:
1. In the introduction, could you briefly mention the everyday use of CO? Does it have any industrial applications?

2. What is the nutritional composition of CO? You only mention the presence of metabolites.

3. If the forage was provided ad libitum, how did you ensure that the feed was a 50:50 forage:concentrate ratio?

4. Can you report the energy content of the diet?

5. How was the time zero shown in Figure 1A obtained (technically)?

6. Please specify the rumen fluid sampling method used for time zero. It is understood that samples were taken every 2 hours to obtain the baseline value (t0)?

7. You mention that fluid samples were taken from different points; could you specify (dorsal sac, ventral sac, strata, etc.)?

Author Response

Authors (AU) reply to Reviewer #1 (R1):

R1: In the introduction, could you briefly mention the everyday use of CO? Does it have any industrial applications?.

1. : Yes, it has been included in the Introduction. The CO has been traditionally used in medicinal, cosmetic, and industrial products because of its analgesic and anti-inflammatory properties. Lines 37-45:

The COP is extracted from the tree copaibeira in Brazil (Copaifera spp.), which is a native tree of tropical regions in Latin America and West Africa that produces an oil-resin ob-tained from the drilling of the trunk [7]. This oil-resin composition varies according to the species, but it commonly contains approximately 80% sesquiterpenes and 20% diterpenes. Among these compounds, β-caryophyllene is the predominant sesquiterpene, represent-ing nearly 50% of the composition of COP [8]. Considering this chemical profile, COP has been traditionally used for centuries due to its analgesic and anti-inflammatory properties and is currently applied in medicinal, cosmetic, and industrial products (Sousa, et al. 2023).

R1: What is the nutritional composition of CO? You only mention the presence of metabolites.

1. : We do not have this information, as copaiba oil can be classified either as an oleoresin or as an essential oil depending on the extraction and processing method. Therefore, only the chemical composition was considered in the present study. Based on the literature, the available data are more specific to the relative concentrations of sesquiterpenes and diterpenes in COP.

R1: If the forage was provided ad libitum, how did you ensure that the feed was a 50:50 forage: concentrate ratio?.

88. :: You are right, I’m sorry for the mistake. This has been corrected, lines: 85-88.

The roughage was supplied ad libitum twice daily at 7:00 a.m. and 5:00 p.m. The amount of roughage offered was adjusted daily to maintain refusals of approximately 150 g kg^-1 BW. The concentrate supplement was supplied in the amount of 15 g kg^-1 BW once a day at 7:00 a.m. in a separate feeder.

R1: Can you report the energy content of the diet?

AU.: Sorry, we can’t estimate this because we don’t have lignin analysis (Weiss). For the estimate, table values ​​for the feedstuffs (corn, soybean meal, NRC, 2000) were considered. The TDN values ​​were 40% and 80.5% for hay and concentrate, respectively.

R1: How was the time zero shown in Figure 1A obtained (technically)?.

130. : The time zero was before the morning feeding as described in lines 121-130.

The DM soluble fraction was determined by placing a sample of hay in a nylon bag and placing bags in water (38°C) for one hour. Following, bags were washed in a washing machine, oven dried, and weighed. The difference between the initial and final weight was considered as the soluble fraction for each experimental period, which corresponds to the value at 0 h in the DM degradation curve.

“The ruminal disappearance procedures were performed as described by Cortada Neto et al. [12].”

R1: Please specify the rumen fluid sampling method used for time zero. It is understood that samples were taken every 2 hours to obtain the baseline value (t0)?.

1. : Thanks! It has been corrected. The time zero was considered the time before feeding. Lines 121-128:

For the determination of VFA's profile, pH, and N-NH3 ruminal fluid was sampled manually via cannula from five different rumen sites (cranial dorsal, cranial ventral, cen-tral, caudal dorsal, and caudal ventral) on the last two days of each experimental period. During this period, samples were taken at four-hour intervals to obtain representative samples. On the first day, samples were collected at 07:00 h (before feeding) and subsequently at 09:00, 13:00, 17:00 h (before feeding), 21:00, 01:00, and 05:00 h. On the second day, samples were collected at 07:00 h (before feeding), 11:00, 15:00, 19:00, 23:00, and 03:00 h. The intercalated sampling schedule between days allowed a final 2-h interval across sampling times throughout the experimental period. The samples before feeding were considered time zero. Sampling was always performed in the same sequence among animals to minimize sampling interference.

 

R1: You mention that fluid samples were taken from different points; could you specify (dorsal sac, ventral sac, strata, etc.)?

1. : This has been included. Lines 121-123:

For the determination of VFA's profile, pH, and N-NH3 ruminal fluid was sampled manually via cannula from five different rumen sites (cranial dorsal, cranial ventral, cen-tral, caudal dorsal, and caudal ventral) on the last two days of each experimental period.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript ID fermentation-4278292

Do different concentrations of copaiba oil (Copaifera spp.) in the diet of steers affect ruminal fermentation?

GENERAL REMARKS

The manuscript evaluates the effects of copaiba oil supplementation on intake, digestibility, ruminal fermentation, and in situ degradability in cattle. The topic is relevant within the context of phytogenic additives as potential alternatives to ionophores in ruminant nutrition. The experimental approach (5 × 5 Latin square with rumen-cannulated animals) is appropriate in principle, and the dataset is technically comprehensive. However, in its current form, the manuscript presents major limitations that weaken the strength and interpretability of the findings. A first critical issue concerns the limited biological response observed across treatments. Most measured variables, including intake, digestibility, degradability, and fermentation parameters, were not significantly affected by copaiba oil supplementation. While negative results are valid, the manuscript does not adequately address whether this lack of response reflects a true absence of biological effect, insufficient statistical power, or suboptimal experimental conditions such as dosage, diet composition, or adaptation period. The absence of this discussion limits the scientific relevance of the study and prevents a clear interpretation of the results. A second major limitation relates to statistical power and experimental sensitivity. The study relies on a small number of animals (n = 5), which is typical for Latin square designs but inherently limits the ability to detect moderate treatment effects. This aspect is not critically discussed. As a result, the manuscript risks Type II error (false negatives), particularly when interpreting the absence of significant differences. The lack of effect should therefore be interpreted cautiously and explicitly framed within these limitations. A third issue concerns the interpretation of results. In several sections, the manuscript attempts to attribute biological meaning to minimal or non-significant differences, or extrapolates potential mechanisms without direct supporting evidence. This creates a mismatch between the strength of the data and the level of inference and leads to overinterpretation despite largely neutral results. Finally, the manuscript is affected by numerous language, formatting, and consistency issues, including grammatical errors, incorrect units, typographical mistakes, and inconsistent terminology. These issues significantly reduce clarity and must be addressed through a thorough revision.

Overall, while the dataset is technically sound, the manuscript requires major revision to improve the interpretation of largely null results, explicitly acknowledge experimental limitations, and correct formal and linguistic issues.

Below are specific comments intended to support a detailed revision of the manuscript.

SPECIFIC COMMENTS

Lines 1–3: The title is formulated as a question, which is not appropriate for a scientific article. It should be rewritten as a declarative statement.

Lines 18–20: Treatment description is inconsistent (“2.50 COP”). Units must be clearly defined (e.g., g kg⁻¹ DM).

Lines 21–26: Redundancy and grammatical issues are present (e.g., “monensin increases”). The authors should use past tense consistently and remove duplicated information.

Lines 25–26: The conclusion is too definitive given the absence of strong effects. Statements should be reformulated using non-causal language (e.g., “no significant effects were observed”).

Lines 32–34: Grammatical errors and incorrect sentence structure are present (“Strategies as phytogenic additives has been used”). This section requires rewriting for clarity and correctness.

Lines 38–40: Statements regarding phytogenic additives are overly general. The authors should better contextualize copaiba oil within the existing literature.

Lines 48–53: There is a conceptual inconsistency between strong antimicrobial claims and statements indicating limited knowledge. This section should be clarified to avoid contradiction.

Lines 53–55: The objective is not clearly formulated. The authors should explicitly state the hypothesis and expected mode of action of copaiba oil. In addition, the manuscript does not clearly define whether the expected response is antimicrobial, fermentation-modulating, or intake-driven. This lack of a mechanistic hypothesis weakens the experimental rationale and should be explicitly addressed.

Lines 62–64: Incorrect phrasing is used (“with age the two years”) and should be corrected. More importantly, no justification of sample size is provided. The authors should explicitly acknowledge that the experimental design (n = 5 in a Latin square) has limited statistical power to detect moderate treatment effects, which directly affects the interpretation of the largely non-significant results.

Lines 73–75: Incorrect units are reported (“2.50 kg⁻¹”) and must be corrected to g kg⁻¹. Furthermore, the choice of inclusion levels is not justified. The authors should provide a clear rationale for the selected doses and discuss whether these levels are expected to elicit a measurable biological response under the given dietary conditions.

Lines 76–77: Reference to literature is made without citation. A supporting reference is required.

Lines 101–105: The digestibility equation is unclear and poorly formatted. It should be rewritten using proper mathematical notation.

Lines 114–116: The degradability model is incorrectly presented (“bxc”). Proper notation (b × c) should be used. Additionally, the authors should clarify how model parameters were estimated and whether goodness-of-fit or residual diagnostics were evaluated.

Lines 121–123: The sampling scheme is unclear and inconsistent with the number of time points reported. The authors should clarify the design.

Lines 155–164: The statistical model lacks critical details. The authors should clearly define fixed and random effects, treatment structure, and the handling of repeated measures if applicable. In particular, it is not specified whether animal and period were treated as random effects in the Latin square model, which is essential for correct variance partitioning. Moreover, potential carry-over effects between periods are not discussed and should be explicitly addressed.

Lines 181–183: There is no mention of multiple testing correction. This should be addressed, particularly given the number of variables analyzed.

Lines 185–210: Results are generally well organized, but the manuscript does not provide sufficient information on variability and effect size. The authors should complement P-values with measures of dispersion and discuss biological relevance. Importantly, the absence of significant differences across most variables should not be interpreted as evidence of no effect. The authors should explicitly discuss the possibility of Type II error and the limitations of statistical power.

Table 2 (associated with Lines 185–190): Formatting inconsistencies are present, including incorrect or missing units and inconsistent superscripts. These must be standardized.

Table 3 (associated with Lines 190–200): Numerical formatting errors are present (e.g., comma vs decimal separator). These require correction.

Figure 1 (associated with Lines 200–205): The caption is unclear and lacks sufficient explanation of variables. It should be revised for clarity.

Figure 2 (associated with Lines 205–210): Axis labels include non-English text (“Título do Eixo”). This must be corrected before publication.

Lines 257–261: The discussion is largely descriptive and does not critically interpret the absence of treatment effects. The authors should explicitly address why no effects were observed. Specifically, the discussion should consider potential explanatory factors such as dietary composition (e.g., forage-to-concentrate ratio), insufficient dosage, adaptation period, or microbial resilience, rather than limiting interpretation to descriptive reporting.

Lines 281–285: An important point regarding diet composition is introduced too late. This should be integrated earlier in the discussion to support interpretation.

Lines 292–294: Grammatical errors are present (“The phytogene additives has shown”) and should be corrected.

Lines 296–299: Interpretation of ruminal pH changes is speculative and should be moderated due to lack of direct supporting evidence. The authors should avoid attributing functional significance to minor or non-significant variations without supporting data on fermentation dynamics.

Lines 323–324: The term “ionospheric effect” is incorrect and should be replaced with “ionophore-like effect”. Additionally, the comparison with monensin should be critically reconsidered, as no clear functional equivalence is demonstrated in the results.

Lines 329–332: The conclusions are too general and do not adequately reflect the largely neutral results. The authors should emphasize the absence of significant effects, acknowledge experimental limitations, and avoid overgeneralization. The conclusions should clearly state that, under the tested conditions, copaiba oil supplementation did not produce measurable effects on the evaluated parameters, rather than implying broader applicability

Comments on the Quality of English Language

The English language is generally understandable and allows the reader to follow the main structure of the manuscript. However, the text requires substantial revision to improve clarity, precision, and overall readability. Several sections contain grammatical errors, incorrect sentence structures, and inconsistent terminology, which reduce the scientific quality of the manuscript. In addition, the writing style is often overly assertive, particularly when interpreting largely non-significant results. A thorough revision by a proficient English speaker or a professional editing service is strongly recommended to improve clarity, coherence, and tone.

Author Response

Authors (AU) reply to Reviewer #2 (R2):

R2: The manuscript evaluates the effects of copaiba oil supplementation on intake, digestibility, ruminal fermentation, and in situ degradability in cattle. The topic is relevant within the context of phytogenic additives as potential alternatives to ionophores in ruminant nutrition. The experimental approach (5 × 5 Latin square with rumen-cannulated animals) is appropriate in principle, and the dataset is technically comprehensive. However, in its current form, the manuscript presents major limitations that weaken the strength and interpretability of the findings.

• A first critical issue concerns the limited biological response observed across treatments. Most measured variables, including intake, digestibility, degradability, and fermentation parameters, were not significantly affected by copaiba oil supplementation. While negative results are valid, the manuscript does not adequately address whether this lack of response reflects a true absence of biological effect, insufficient statistical power, or suboptimal experimental conditions such as dosage, diet composition, or adaptation period. The absence of this discussion limits the scientific relevance of the study and prevents a clear interpretation of the results.

AU: We appreciate the reviewer’s comment. The suggested discussion was incorporated into the manuscript. In addition, we emphasize that the experimental conditions, including diet composition, adaptation period, and dosage, were carefully designed based on previous studies, supporting the reliability of the observed responses.

• A second major limitation relates to statistical power and experimental sensitivity. The study relies on a small number of animals (n = 5), which is typical for Latin square designs but inherently limits the ability to detect moderate treatment effects. This aspect is not critically discussed. As a result, the manuscript risks Type II error (false negatives), particularly when interpreting the absence of significant differences. The lack of effect should therefore be interpreted cautiously and explicitly framed within these limitations.

AU: We appreciate the reviewer’s consideration regarding statistical power. The discussion was revised to clarify that the 5 × 5 Latin square design is commonly adopted in studies with rumen-cannulated animals, as it allows control of animal and period effects, increasing experimental precision while reducing the number of animals required. Nevertheless, the interpretation of non-significant effects was revised and discussed cautiously, considering the possibility of Type II error.

 

• A third issue concerns the interpretation of results. In several sections, the manuscript attempts to attribute biological meaning to minimal or non-significant differences, or extrapolates potential mechanisms without direct supporting evidence. This creates a mismatch between the strength of the data and the level of inference and leads to overinterpretation despite largely neutral results. Finally, the manuscript is affected by numerous language, formatting, and consistency issues, including grammatical errors, incorrect units, typographical mistakes, and inconsistent terminology. These issues significantly reduce clarity and must be addressed through a thorough revision.

AU: We sincerely appreciate the reviewer’s careful evaluation and constructive comments, which substantially contributed to improving the manuscript. The discussion and interpretation of the results were revised to avoid overinterpretation and to better align the conclusions with the observed data. In addition, the manuscript underwent a thorough revision regarding language, formatting, terminology, and consistency issues, and all suggested corrections were carefully addressed.

Overall, while the dataset is technically sound, the manuscript requires major revision to improve the interpretation of largely null results, explicitly acknowledge experimental limitations, and correct formal and linguistic issues.

 

Below are specific comments intended to support a detailed revision of the manuscript.

AU: We sincerely thank you for the evaluation and all the suggestions provided to the manuscript. The comments were extremely valuable and substantially improved the clarity, scientific rigor, and overall quality of our study. We truly appreciate the time and effort dedicated to reviewing this draft.

 

R2:  The title is formulated as a question, which is not appropriate for a scientific article. It should be rewritten as a declarative statement.

AU: Thanks. This has been corrected. Line 1-3:

Different concentrations of copaiba oil (Copaifera spp.) in the ruminal fermentation

R2: Lines 18–20: Treatment description is inconsistent (“2.50 COP”). Units must be clearly defined (e.g., g kg⁻¹ DM)..

AU: Thanks. This has been corrected. Line 19-20:

Control – (0 g kg^-1 of COP), 1.25 g kg^-1 COP, 2.50 g kg^-1 COP, and 3.75 g kg^-1 COP dry matter (DM),

 

R2: Lines 21–26: Redundancy and grammatical issues are present (e.g., “monensin increases”). The authors should use past tense consistently and remove duplicated information.

AU: Thanks for the suggestion. We apologize for the mistakes. This has been corrected. Line 24-25:

In contrast, monensin increased (P ≤ 0.05) the concentrations of NH3-N and propionate (mmol L-1).

R2: Lines 25–26: The conclusion is too definitive given the absence of strong effects. Statements should be reformulated using non-causal language (e.g., “no significant effects were observed”).

AU:  Thanks! This has been corrected. Lines 25-27:

Overall, COP supplementation did not modify ruminal fermentation under the conditions of this study. However, further studies are needed to assess its effects in diets with higher forage proportions, which better represent grazing systems.

 

R2: Lines 32–34: Grammatical errors and incorrect sentence structure are present (“Strategies as phytogenic additives has been used”). This section requires rewriting for clarity and correctness.

AU: Thanks! This section has been rewritten. Lines 32-57.

 

R2: Lines 38–40: Statements regarding phytogenic additives are overly general. The authors should better contextualize copaiba oil within the existing literature.

AU: Thanks! This section has been rewritten. Lines 32-57.

R2: Lines 48–53: There is a conceptual inconsistency between strong antimicrobial claims and statements indicating limited knowledge. This section should be clarified to avoid contradiction.

AU:  Thanks! This section has been rewritten. Lines 32-57.

 

R2: Lines 53–55: The objective is not clearly formulated. The authors should explicitly state the hypothesis and expected mode of action of copaiba oil. In addition, the manuscript does not clearly define whether the expected response is antimicrobial, fermentation-modulating, or intake-driven. This lack of a mechanistic hypothesis weakens the experimental rationale and should be explicitly addressed.

AU: Thanks, the objective has been corrected, and the hypothesis has been included. Lines 52-61:

This antimicrobial activity of COP suggests its potential use as a feed additive in ruminant diets. However, its application as a nutritional additive, particularly for manipulation of the rumen environment, remains recent, and its effects are not yet well established. Therefore, the objective of this study was to evaluate the effects of different dietary concentrations of COP on intake, nutrient digestibility, in situ ruminal degradability, and ruminal fermentation variables (pH, N-NH3, and VFAs) in steers. We hypothesized that the sesquiterpenes and diterpenes present in COP would selectively modulate ruminal microbial populations, promoting changes in ruminal fermentation patterns, particularly in VFA production and ammonia concentration, while maintaining nutrient intake and digestibility in steers fed increasing concentrations of COP.

R2: Lines 62–64: Incorrect phrasing is used (“with age the two years”) and should be corrected. More importantly, no justification of sample size is provided. The authors should explicitly acknowledge that the experimental design (n = 5 in a Latin square) has limited statistical power to detect moderate treatment effects, which directly affects the interpretation of the largely non-significant results.

AU: Thanks. This sentence has been corrected, and the explanations regarding the small sample size have been included in the discussion section. Line 68-70:

Five rumen-cannulated crossbred steers (1/2 Nellore x 1/2 Holstein), approximately 2 years old and with an initial body weight (BW) of 279 ± 22 kg, were assigned to a 5 x 5 Latin square design.

AU: We appreciate the reviewer’s comment and agree that the interpretation of non-significant results should be made cautiously. However, we also emphasize that Latin square designs with a limited number of animals are commonly used in studies with rumen-cannulated cattle, as this approach allows effective control of animal and period variation while reducing animal use and increasing experimental control.

R2: Lines 73–75: Incorrect units are reported (“2.50 kg⁻¹”) and must be corrected to g kg⁻¹. Furthermore, the choice of inclusion levels is not justified. The authors should provide a clear rationale for the selected doses and discuss whether these levels are expected to elicit a measurable biological response under the given dietary conditions.

AU: This has been corrected. Line 76:

control (0 g of COP), COP 1.25 (1.25 g kg-1 of COP), COP 2.50 (2.50 g kg-1 of COP), and COP 3.75 (3.75 g kg-1 of COP) per kg of DM of the total diet, respectively.

R2: Lines 76–77: Reference to literature is made without citation. A supporting reference is required.

AU: Thanks! We excluded this sentence and discussed the concentrations of the copaiba oil in the discussion section.

R2: Lines 101–105: The digestibility equation is unclear and poorly formatted. It should be rewritten using proper mathematical notation.

AU: Thanks! This has been corrected. Lines 104-105:

Nutrient’s intake (amount offered - refusal) and total tract apparent digestibility coefficients [(intake - output in feces)/ intake] of dry matter (DM)

 

R2: Lines 114–116: The degradability model is incorrectly presented (“bxc”). Proper notation (b × c) should be used. Additionally, the authors should clarify how model parameters were estimated and whether goodness-of-fit or residual diagnostics were evaluated.

AU: Thanks! This has been corrected. Lines 119-120:

based on the equation: ED = a + (b x c)/(c + k)

 

R2: Lines 121–123: The sampling scheme is unclear and inconsistent with the number of time points reported. The authors should clarify the design.

AU: Thanks! This has been corrected. Lines 125-135:

For the determination of VFA's profile, pH, and N-NH3 ruminal fluid was sampled manually via cannula from five different rumen sites (cranial dorsal, cranial ventral, cen-tral, caudal dorsal, and caudal ventral) on the last two days of each experimental period. During this period, samples were taken at four-hour intervals to obtain representative samples. On the first day, samples were collected at 07:00 h (before feeding) and subse-quently at 09:00, 13:00, 17:00 h (before feeding), 21:00, 01:00, and 05:00 h. On the second day, samples were collected at 07:00 h (before feeding), 11:00, 15:00, 19:00, 23:00, and 03:00 h. The intercalated sampling schedule between days allowed a final 2-h interval across sampling times throughout the experimental period. The samples before feeding were considered time zero. Sampling was always performed in the same sequence among an-imals to minimize sampling interference.

 

R2: Lines 155–164: The statistical model lacks critical details. The authors should clearly define fixed and random effects, treatment structure, and the handling of repeated measures if applicable. In particular, it is not specified whether animal and period were treated as random effects in the Latin square model, which is essential for correct variance partitioning. Moreover, potential carry-over effects between periods are not discussed and should be explicitly addressed.

AU: We appreciate the reviewer’s comments and suggestions, which contributed to improving the description of the statistical analyses. The statistical procedures were revised and described in greater detail in the manuscript. For intake and digestibility variables, treatment was considered a fixed effect, while animal and period were included as random effects in the Latin square model. For variables evaluated over time, time and the treatment × time interaction were also considered fixed effects. In addition, the adaptation period adopted between experimental periods was designed to minimize potential carry-over effects.

R2: Lines 181–183: There is no mention of multiple testing correction. This should be addressed, particularly given the number of variables analyzed.

AU: We appreciate the reviewer’s observation. The statistical analysis section was revised to clarify that repeated measures were analyzed using covariance structures selected according to the lowest Akaike Information Criterion (AIC). In addition, when significant effects were detected by ANOVA, mean comparisons were adjusted using Tukey’s test. As commonly adopted in animal nutrition studies, each response variable was analyzed and interpreted independently.

R2: Lines 185–210: Results are generally well organized, but the manuscript does not provide sufficient information on variability and effect size. The authors should complement P-values with measures of dispersion and discuss biological relevance. Importantly, the absence of significant differences across most variables should not be interpreted as evidence of no effect. The authors should explicitly discuss the possibility of Type II error and the limitations of statistical power.

AU: We appreciate the reviewer’s important comments and suggestions. The results and discussion sections were revised to improve data interpretation, including a more cautious discussion regarding the biological relevance of the observed responses. In addition, measures of variability were clarified throughout the manuscript to improve the presentation and interpretation of the results.

R2: Table 2 (associated with Lines 185–190): Formatting inconsistencies are present, including incorrect or missing units and inconsistent superscripts. These must be standardized.

AU: Thanks for the suggestion. I did not find the missing units. Could you point it out?

R2: Table 3 (associated with Lines 190–200): Numerical formatting errors are present (e.g., comma vs decimal separator). These require correction.

AU: Thanks for the suggestion. This has been corrected. Lines 216-217.

R2: Figure 1 (associated with Lines 200–205): The caption is unclear and lacks sufficient explanation of variables. It should be revised for clarity.

AU: Thanks for the suggestion. This has been corrected. Line 221.

Figure 1. Hay DM degradation (g 100 g-1) in the rumen of steers receiving different concentrations of copaiba oil (COP). (a) Vertical bars represent the standard deviation. An asterisk (*) indicates differences among treatments at a given time (P ≤ 0.05). Fraction “a”: 23.16 g 100 g-1. (b) Vertical bars represent the standard deviation.

 

R2: Figure 2 (associated with Lines 205–210): Axis labels include non-English text (“Título do Eixo”). This must be corrected before publication.

AU: Thank you for the correction.

R2: Lines 257–261: The discussion is largely descriptive and does not critically interpret the absence of treatment effects. The authors should explicitly address why no effects were observed. Specifically, the discussion should consider potential explanatory factors such as dietary composition (e.g., forage-to-concentrate ratio), insufficient dosage, adaptation period, or microbial resilience, rather than limiting interpretation to descriptive reporting.

AU: Thanks for the suggestion. We improved the discussion in an attempt to attend this suggestion.

R2: Lines 281–285: An important point regarding diet composition is introduced too late. This should be integrated earlier in the discussion to support interpretation.

AU: Thanks for the suggestion. It has been changed to the beginning of the discussion. Lines 273-280:

The lack of effects of COP on changes in nutrient intake corroborates results from other studies evaluating the addition of phytogenic additives to the diet of ruminants [21-23]. However, the use of COP as nutritional additives has shown different results on feed intake, which may be associated with high inclusion variability and the composition of the evaluated compounds [24]. It's important to highlight that the diet composition, such as the high roughage:concentrate ratio (approximately 50:50), low forage quality (low crude protein and high NDF), and low supplement intake by the animal, may have partially explained the lack of effects observed in the current study.

 

R2: Lines 292–294: Grammatical errors are present (“The phytogene additives has shown”) and should be corrected.

AU: Thanks for the suggestion. This has been corrected!

R2: Lines 296–299: Interpretation of ruminal pH changes is speculative and should be moderated due to lack of direct supporting evidence. The authors should avoid attributing functional significance to minor or non-significant variations without supporting data on fermentation dynamics.

AU: Thanks for the suggestion. This has been corrected! Lines 350-356:

At higher inclusion levels, responses on rumen fermentation are often inconsistent and may reflect non-linear dose-response relationships, microbial adaptation to bioactive compounds, or interactions with diet composition [40]. In the present study, the rela-tively high COP levels of inclusion in the diet, combined with a high-forage diet and moderate rumen pH, may have contributed to a reduced sensitivity of the rumen mi-crobial ecosystem to phytogenic modulation, which could partly explain the lack of ef-fects observed in this study.

 

R2: Lines 323–324: The term “ionospheric effect” is incorrect and should be replaced with “ionophore-like effect”. Additionally, the comparison with monensin should be critically reconsidered, as no clear functional equivalence is demonstrated in the results.

AU: Thanks for the suggestion. This has been corrected! Lines 365-370:

Copaiba oil (Copaifera spp.) supplementation (1.25 to 3.75) did not produce meas-urable effects on feed intake, nutrient digestibility, ruminal degradation and fermenta-tion parameters of steers under the conditions of this study. In contrast, monensin re-duced forage intake and fiber degradation and a shift of ruminal fermentation, pro-ducing higher concentration of propionate. Further studies are recommended to eluci-date the effects of COP supplementation on steer metabolism and performance.

 

R2: Lines 329–332: The conclusions are too general and do not adequately reflect the largely neutral results. The authors should emphasize the absence of significant effects, acknowledge experimental limitations, and avoid overgeneralization. The conclusions should clearly state that, under the tested conditions, copaiba oil supplementation did not produce measurable effects on the evaluated parameters, rather than implying broader applicability

AU: Thanks for the suggestion. This has been corrected! Lines

 

R2: Comments on the Quality of English Language

The English language is generally understandable and allows the reader to follow the main structure of the manuscript. However, the text requires substantial revision to improve clarity, precision, and overall readability. Several sections contain grammatical errors, incorrect sentence structures, and inconsistent terminology, which reduce the scientific quality of the manuscript. In addition, the writing style is often overly assertive, particularly when interpreting largely non-significant results. A thorough revision by a proficient English speaker or a professional editing service is strongly recommended to improve clarity, coherence, and tone.

AU: Thanks for the suggestion. This has been corrected!

Thanks for your contribution.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has improved substantially compared with the previous version. The authors have addressed several major concerns, particularly by reformulating the title, clarifying the treatment description and units, improving the objective and hypothesis, expanding the description of the ruminal sampling scheme, and moderating the interpretation of the largely non-significant effects of copaiba oil. The Abstract clearly states that copaiba oil supplementation did not modify ruminal fermentation under the tested conditions, while also indicating the need for further studies in more representative dietary contexts. The Discussion has also been improved. The authors now explicitly consider diet composition, forage-to-concentrate ratio, low forage quality, and low supplement intake as possible explanations for the lack of response to copaiba oil. This addition directly addresses one of the main previous concerns regarding the biological interpretation of null results. The revised text also avoids presenting copaiba oil as having an ionophore-like effect and more clearly distinguishes its lack of measurable effects from the expected response to monensin.

However, some aspects remain only partially resolved. The issue of limited statistical power is acknowledged in the authors’ response, but it is not sufficiently visible as a clear limitation in the manuscript itself. The authors explain that the 5 × 5 Latin square design is commonly used in rumen-cannulated cattle studies and that it controls animal and period effects, but the manuscript should still explicitly state that the small number of animals may limit the detection of moderate treatment effects and that non-significant results should therefore be interpreted cautiously. Several formal and language issues also remain.

Overall, the authors have satisfactorily addressed the main scientific concerns, especially regarding overinterpretation and the framing of the largely neutral results. The manuscript is now much closer to publication, but minor revision is still required to correct residual editing errors, make the limitation related to statistical power explicit in the manuscript, and ensure consistency of tables, figures, units, and language.

Comments on the Quality of English Language

The English language has improved substantially compared with the previous version, and the manuscript is now generally clear and understandable. Nevertheless, some grammatical inaccuracies, awkward sentence constructions, and stylistic inconsistencies are still present throughout the text. A final careful linguistic revision by a fluent English speaker or professional editing service is recommended to improve readability and ensure overall language consistency before publication.

Author Response

Authors (AU) reply to Reviewer #2 (R2):

R2: Comments and Suggestions for Authors

The revised manuscript has improved substantially compared with the previous version. The authors have addressed several major concerns, particularly by reformulating the title, clarifying the treatment description and units, improving the objective and hypothesis, expanding the description of the ruminal sampling scheme, and moderating the interpretation of the largely non-significant effects of copaiba oil. The Abstract clearly states that copaiba oil supplementation did not modify ruminal fermentation under the tested conditions, while also indicating the need for further studies in more representative dietary contexts. The Discussion has also been improved. The authors now explicitly consider diet composition, forage-to-concentrate ratio, low forage quality, and low supplement intake as possible explanations for the lack of response to copaiba oil. This addition directly addresses one of the main previous concerns regarding the biological interpretation of null results. The revised text also avoids presenting copaiba oil as having an ionophore-like effect and more clearly distinguishes its lack of measurable effects from the expected response to monensin.

However, some aspects remain only partially resolved. The issue of limited statistical power is acknowledged in the authors’ response, but it is not sufficiently visible as a clear limitation in the manuscript itself. The authors explain that the 5 × 5 Latin square design is commonly used in rumen-cannulated cattle studies and that it controls animal and period effects, but the manuscript should still explicitly state that the small number of animals may limit the detection of moderate treatment effects and that non-significant results should therefore be interpreted cautiously. Several formal and language issues also remain.

1. : Thanks for all your suggestions. This has been included now. Lines 356-365:

Although the 5 × 5 Latin square design commonly used in rumen-cannulated cattle studies provides substantial experimental control by accounting for animal and period effects, caution is warranted when extrapolating the present findings to broader pro-duction conditions. Within the experimental conditions adopted, COP supplementation did not affect the evaluated metabolic parameters. Nevertheless, additional studies involving larger larger numbers of animals and long-term performance responses may contribute to a more comprehensive understanding of the biological relevance of COP supplementation in ruminant diets.

Overall, the authors have satisfactorily addressed the main scientific concerns, especially regarding overinterpretation and the framing of the largely neutral results. The manuscript is now much closer to publication, but minor revision is still required to correct residual editing errors, make the limitation related to statistical power explicit in the manuscript, and ensure consistency of tables, figures, units, and language.

1. : The English has been revised by an American native speaker. We hope the language is better now.

 

R2: Comments on the Quality of English Language

The English language has improved substantially compared with the previous version, and the manuscript is now generally clear and understandable. Nevertheless, some grammatical inaccuracies, awkward sentence constructions, and stylistic inconsistencies are still present throughout the text. A final careful linguistic revision by a fluent English speaker or professional editing service is recommended to improve readability and ensure overall language consistency before publication.

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AU: Thanks for the suggestion. This has been corrected!

Thanks for your contribution.