Effect of Dietary Inclusion of Brown Seaweed (Macrocystis pyrifera) on Oxidative Stress of Grass-Fed Dairy Cows

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comment sheet is attached 

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Manuscript entitled “Effect of the inclusion of brown seaweed (Macrocystis pyrifera) in the diet of grass-fed dairy cows on oxidative stress” is very good topic and need of hour. But there are some points which needs clarification

Abstract:

Line # 15 please write the full name of M. Pyrifera.

Thank you. We have corrected the text

Introduction:

Write the hypothesis and objective of the current study clearly.

Thank you very much for your comment. We have clarified our assumptions and objectives in lines 86-91.

Material and method:

Please clarify, how sea weed was fed either mix with commercial concentrate or fed separately.  

Seaweed and concentrate were fed at the same time. It was clarified in the text as suggested.

Further, Table 2 the CP value of pasture is 22 %, while for commercial concentrate 11.20 %, Please check. if possible, give the composition of commercial concentrate for more clarification.

It was included the composition of commercial concentrate as suggested.

 Please give amount of DMI of the animals in different group. As seaweeds reported to have negative effect on the DMI of the animals, which is also evident from the Table 1 and Table 2.  We have included the amount of DMI of the animals in different group as suggested.

Statistical analysis: Repeated measures analysis can be performed. However, as I am not a statistician, input from a statistician would be very helpful in this regard. 

We acknowledge the feedback and have rectified the error in the "statistical analysis" section. In our paper, we conducted an analysis using repeated measures ANOVA with post-hoc comparisons. Additionally, we had initially employed a mixed regression model, using the cow as a random effect to account for the error in each repetition (lines 185-186 /188-189)

Reference:

Please check the reference style and follow general guidelines.

Thank you very much. We made the changes in the reference formats 

Line 297# Delete 2017 as it is repeated twice.

Thank you. We have corrected the reference (Line 380)

Reference no. 19 year is not provided.

We have corrected the reference (Line 416)

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript investigates the effect of incorporating brown seaweed (Macrocystis pyrifera) into the diet of grass-fed dairy cows on oxidative stress. The study aims to evaluate the potential antioxidant benefits of M. pyrifera when included in the diet at two different levels. The research is timely and relevant, considering the growing interest in natural feed additives for improving animal health and productivity. The manuscript is well-structured, with a clear objective, methodology, results, and discussion sections. However, some areas need clarification and additional analysis to strengthen the study's findings.

1. The introduction provides a good overview of oxidative stress in dairy cows and the potential antioxidant properties of brown seaweed. However, it could benefit from a more detailed explanation of how M. pyrifera has been previously used in animal nutrition, highlighting any gaps in knowledge that this study aims to address.

2. Introduction: The manuscript could include more recent studies on the use of seaweed in animal nutrition, particularly focusing on studies that have demonstrated significant effects on oxidative stress and antioxidant status.

3. The manuscript could include more recent studies on the use of seaweed in animal nutrition, particularly focusing on studies that have demonstrated significant effects on oxidative stress and antioxidant status.

4. Include more detailed information about the chemical composition of M. pyrifera used in this study. This would help understand the potential variations in the results and the specific components responsible for the observed effects.

5. Explain how the actual intake of M. pyrifera by cows was monitored throughout the study. It is important to confirm that cows consumed the intended amount of seaweed.

6. Please clarify why blood samples were collected specifically on days 18, 45, and 61. Justify the choice of these time points for evaluating oxidative stress markers.

7. The manuscript mentions using the ratio between MDA and TEAC to calculate OSi. Discuss the validity and relevance of this index in the context of evaluating oxidative stress in dairy cows.

8. Expand on the discussion to compare the results of this study with other similar studies. Discuss why the inclusion of M. pyrifera did not significantly affect MDA, TEAC, and OSi, and propose potential mechanisms.

 

9. Suggest directions for future research, such as investigating the long-term effects of M. pyrifera inclusion, potential interactions with other dietary components, and the exploration of different seaweed species or processing methods.

Author Response

This manuscript investigates the effect of incorporating brown seaweed (Macrocystis pyrifera) into the diet of grass-fed dairy cows on oxidative stress. The study aims to evaluate the potential antioxidant benefits of M. pyrifera when included in the diet at two different levels. The research is timely and relevant, considering the growing interest in natural feed additives for improving animal health and productivity. The manuscript is well-structured, with a clear objective, methodology, results, and discussion sections. However, some areas need clarification and additional analysis to strengthen the study's findings.

1. The introduction provides a good overview of oxidative stress in dairy cows and the potential antioxidant properties of brown seaweed. However, it could benefit from a more detailed explanation of how M. pyrifera has been previously used in animal nutrition, highlighting any gaps in knowledge that this study aims to address.

Thank you very much for your comment. We have enhanced the introduction section by incorporating various articles discussing the impact of seaweed inclusion in the nutrition of diverse production systems. (lines 59-72)

2. Introduction: The manuscript could include more recent studies on the use of seaweed in animal nutrition, particularly focusing on studies that have demonstrated significant effects on oxidative stress and antioxidant status.

Thank you very much for your comment. We have incorporated into the introduction section recent articles on the effects of algae inclusion on oxidative stress and antioxidant status in different production systems (lines 74-86).

3. The manuscript could include more recent studies on the use of seaweed in animal nutrition, particularly focusing on studies that have demonstrated significant effects on oxidative stress and antioxidant status.

Thank you very much for your comment. We have incorporated into the introduction section recent articles on the effects of algae inclusion on oxidative stress and antioxidant status in different production systems (lines 74-86).

4. Include more detailed information about the chemical composition of M. pyrifera used in this study. This would help understand the potential variations in the results and the specific components responsible for the observed effects.

Unfortunately, we don´t have more details about the chemical composition of M. pyrifera.

5. Explain how the actual intake of M. pyrifera by cows was monitored throughout the study. It is important to confirm that cows consumed the intended amount of seaweed.

Explanation how the actual intake of M. pyrifera was monitored throughout the study was included as suggested.

6. Please clarify why blood samples were collected specifically on days 18, 45, and 61. Justify the choice of these time points for evaluating oxidative stress markers.

Blood samples were collected on days 18, 45, and 61, corresponding to three sampling periods throughout the experiment. This study forms part of a larger research project focused on environmental, ruminal, and productive parameters. The work presented here represents a supplementary investigation within the broader experiment. It was included in the text as suggested.

7. The manuscript mentions using the ratio between MDA and TEAC to calculate OSi. Discuss the validity and relevance of this index in the context of evaluating oxidative stress in dairy cows.

We have highlighted the importance of OSI in the evaluation of the oxidative status of cows. (Lines 328 – 333)

8. Expand on the discussion to compare the results of this study with other similar studies. Discuss why the inclusion of M. pyrifera did not significantly affect MDA, TEAC, and OSi, and propose potential mechanisms.

We appreciate your comment as it helps us to delve deeper into certain points of the discussion. We have expanded our discussion of MDA, TEAC, and OSI between lines 274-284, 312-316

9. Suggest directions for future research, such as investigating the long-term effects of M. pyrifera inclusion, potential interactions with other dietary components, and the exploration of different seaweed species or processing methods.

At the end of our discussion, we have included future research projections regarding the use of seaweed and oxidative stress. This takes into consideration factors such as effective doses, interactions with other seaweeds, and methods of extracting compounds with biological activity (Lines 340-350)

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editor and Authors

In this study, the authors aimed to evaluate the effects of different inclusion levels of Macrocystis pyrifera (brown seaweed) in the diets of mid-lactating cows on blood metabolites in relation to oxidative stress, specifically malondialdehyde, Trolox equivalent antioxidant capacity, and oxidant status index.

Specific Comments:

L2: I suggest the title should be rephrased for more clarity

L 35-36: DNA damage caused by free radicals is typically referred to as oxidative damage rather than oxidation. You should revise this sentence.

L 50-53: The phrase “which have been shown to enhance antioxidant mechanisms in vitro experiments” is somewhat vague. It would be more precise to mention that these have been shown to “exhibit antioxidant activity” in vitro rather than “enhance antioxidant mechanisms.” Moreover, the cited article reported antioxidant capacity. 

L62: ‘no studies have evaluated the antioxidant potential of brown algae…’is sounds contradictory considering previous sentences. It is better stated as ‘no studies have evaluated the antioxidant potential of other brown algae, such as M. pyrifera…’

L64: ‘murrah buffalo’ not ‘buffalo murrah’. The entire manuscript should be checked for grammatical accuracy.

L 67: M. pyrifera not M. Pyrifera. Correct across the manuscript.

L77: It should be ‘Data not published’

L88: Use isoenergetic and isoproteic to clarify that the experimental diets had equal energy and protein content

L95-96: by the phrase ‘according to the treatment’, were cows in different treatment groups grazed at different timings or at the same time?

L102: Correct ‘and the dried at 60°C’

L104: Is there any specific reason why the samples were collected midway through the experiment? This can be clarified to avoid ambiguity.

L110: Bromatological test? From Table 2, I presume this is simply the nutritional or chemical composition of the feed. Please include the details of the analysis for clarity.

L119: from all cows

L122-123 Revise the sentence ‘where were centrifuged?’

L127: correct to ‘thiobarbituric acid’

L133: The ratio between pro-oxidant and antioxidant levels is referred to as the oxidant status index (OSi), but the term OSi is not widely used in the same context in scientific literature. Instead, it’s more common to call this the oxidative stress index. Revise this for clarity.

L142: Why is cow considered a random effect? Given that the cows are repeatedly measured over time, this should be clarified as a repeated measures design rather than simply a random effect.

L144: Did the authors carry out a post hoc test? There is no mention of multiple pairwise comparisons.

General Comment: The study is relevant as it explores the potential of incorporating seaweed as a dietary supplement to improve antioxidant status and mitigate oxidative stress in dairy cows, which can influence their health and productivity. However, the study lacks sufficient data, and there are several missen pieces. Examining only MDA, TEAC, and OSi provides a limited understanding of the effect of Macrocystis pyrifera on oxidative stress status in cows. Also, the entire manuscript would benefit from language editing to improve its quality.

 

 

 

 

 

 

 

 

 

 

 

Comments on the Quality of English Language

The english language of the manuscript must be revised to improve its quality.

Author Response

Dear Editor and Authors

In this study, the authors aimed to evaluate the effects of different inclusion levels of Macrocystis pyrifera (brown seaweed) in the diets of mid-lactating cows on blood metabolites in relation to oxidative stress, specifically malondialdehyde, Trolox equivalent antioxidant capacity, and oxidant status index.

Specific Comments:

L2: I suggest the title should be rephrased for more clarity

Thank you very much for the suggestion. We have rephrased the title: “Effect of dietary inclusion of brown seaweed (Macrocystis pyrifera) on oxidative stress of grass-fed dairy cows”

L 35-36: DNA damage caused by free radicals is typically referred to as oxidative damage rather than oxidation. You should revise this sentence.

We have revised the sentence and made changes to it. “This imbalance causes oxidative damage on proteins, DNA, and lipids, followed by tissue damage, which, in turn, produces more free radicals that can lead to further oxidative stress, creating a cycle that is pathological for the animal” (L 35-36)

Thank you for your feedback.

L 50-53: The phrase “which have been shown to enhance antioxidant mechanisms in vitro experiments” is somewhat vague. It would be more precise to mention that these have been shown to “exhibit antioxidant activity” in vitro rather than “enhance antioxidant mechanisms.” Moreover, the cited article reported antioxidant capacity.

Thank you. We have specified the phrase: “ These compounds include phlorotannins, alpha-tocopherol, ascorbic acid, carotenoids, amino acids, peptides, proteins, and flavonoids, which have been shown to enhance antioxidant capacity in vitro experiments using brown and green seaweeds (L 50-53)

L62: ‘no studies have evaluated the antioxidant potential of brown algae…’is sounds contradictory considering previous sentences. It is better stated as ‘no studies have evaluated the antioxidant potential of other brown algae, such as M. pyrifera…’

We have reworded the entire paragraph, including new background, as suggested (Lines 59-73)

L64: ‘murrah buffalo’ not ‘buffalo murrah’. The entire manuscript should be checked for grammatical accuracy.

Thank you. We have corrected the text (Line 88)

L 67: M. pyrifera not M. Pyrifera. Correct across the manuscript.

We regret the error and have corrected the spelling of “M. pyrifera” throughout the text."

L77: It should be ‘Data not published’

Thank you. We have corrected the text (Line 106)

L88: Use isoenergetic and isoproteic to clarify that the experimental diets had equal energy and protein content

Text was edited as suggested.

L95-96: by the phrase ‘according to the treatment’, were cows in different treatment groups grazed at different timings or at the same time?

All treatments were allocated within the same paddock, separated by an electric fence.It was included in the text as suggested.

 

L102: Correct ‘and the dried at 60°C’

Thank you. We have corrected the text. (Line 149)

 

L104: Is there any specific reason why the samples were collected midway through the experiment? This can be clarified to avoid ambiguity.

Samples were collected following a 21-day adaptation period, with the first sampling conducted in week 4, and subsequent samplings in weeks 6 and 9. This study forms part of a larger research project focused on environmental, ruminal, and productive parameters. The work presented here represents a supplementary investigation within the broader experiment. It was included in the text as suggested.

 

L110: Bromatological test? From Table 2, I presume this is simply the nutritional or chemical composition of the feed. Please include the details of the analysis for clarity.

It was a simple chemical analysis of the feed, thank you. Details of the analysis was included as suggested.

L119: from all cows

Thank you. We have corrected the text. (Line 174)

 

L122-123 Revise the sentence ‘where were centrifuged?’

Thank you. We have corrected the text (Lines 179-180): Upon arrival, they were centrifuged at 800g for 20 minutes using a refrigerated centrifuge (Heraeus Varifuge RF)

L127: correct to ‘thiobarbituric acid’

Thank you. We have corrected the text. (Line 183)

 

L133: The ratio between pro-oxidant and antioxidant levels is referred to as the oxidant status index (OSi), but the term OSi is not widely used in the same context in scientific literature. Instead, it’s more common to call this the oxidative stress index. Revise this for clarity.

It was revised as suggested by reviewer.

 

L142: Why is cow considered a random effect? Given that the cows are repeatedly measured over time, this should be clarified as a repeated measures design rather than simply a random effect.

We acknowledge the feedback and have rectified the error in the "statistical analysis" section. In our paper, we conducted an analysis using repeated measures ANOVA with post-hoc comparisons. Additionally, we had initially employed a mixed regression model, using the cow as a random effect to account for the error in each repeated measures (Lines 197-198)

L144: Did the authors carry out a post hoc test? There is no mention of multiple pairwise comparisons.

We acknowledge the feedback and have rectified the error in the "statistical analysis" section. In our paper, we conducted an analysis using repeated measures ANOVA with post-hoc comparisons (Tukey’s HSD) (Lines 200-201)

General Comment: The study is relevant as it explores the potential of incorporating seaweed as a dietary supplement to improve antioxidant status and mitigate oxidative stress in dairy cows, which can influence their health and productivity. However, the study lacks sufficient data, and there are several missen pieces. Examining only MDA, TEAC, and OSi provides a limited understanding of the effect of Macrocystis pyrifera on oxidative stress status in cows. Also, the entire manuscript would benefit from language editing to improve its quality.

We appreciate your comments and suggestions, as they have helped us to delve deeper into ideas and review concepts more thoroughly. We recognize the limitations of the study, particularly those related to the number of oxidative stress indicators measured. Nevertheless, we are confident that this study was conducted rigorously and adds to our understanding of the impact of seaweed use on oxidative stress in dairy cattle, a topic that has been insufficiently explored. Furthermore, this study is intended to serve as a foundation for future research that incorporates a broader range of indicators, seaweed mixtures, and alternative extraction methods.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editors,

 The authors have revised the manuscript rigorously and addressed every comment raised.