Effect of Feeding Sugarcane Bagasse-Extracted Polyphenolic Mixture on the Growth Performance, Meat Quality, and Oxidative and Inflammatory Status of Chronic Heat-Stressed Broiler Chickens

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

“Bagasse-extracted polyphenolic mixture attenuates the growth performance, meat quality, and oxidative and inflammatory status of chronic heat-stressed broiler chickens”

Line 19-21 “Plasma lipid peroxidation content, inflammatory cytokines [interleukin (IL)-6, IL-β], corticosterone, and uric acid concentrations were significantly increased by HS, while the increases were attenuated by BEPM supplementation (P < 0.05)” in the Introduction, there is not enough review about infalmmatory, and metabolism in blood, which can’t supply the evidence of study Plasma lipid peroxidation content, inflammatory cytokines. At least, the review should add the enough information of stress on metabolism and infalmmatory in digestive organs.

Line35-38 “Broiler chickens raised under high-temperature conditions exhibit growth retardation and deterioration of their health, which is termed as heat stress (HS). Increased oxidative stress and inflammatory responses are the hallmarks observed in HS-treated chickens” the concept of heat stress isn’t right. stress is the sum of defence mechanisms or non- specific responses of an organism when it is faced with abnormal situation or extreme demands (McEwen & Akil, 2020; Sahin et al., 2009). see Broiler production challenges in the tropics: A review/Vet Med Sci. 2021;7:831–842.

Heat stress can not equal to oxidative stress. Heat stress is one kinds of very strong oxidative stress.

In recently years, many published papers about heat stress of broiers.

Saracila, M.; Panaite, T.D.; Papuc, C.P.; Criste, R.D. Heat Stress in Broiler Chickens and the Effect of Dietary Polyphenols, with Special Reference to Willow (Salix spp.) Bark Supplements—A Review. Antioxidants 2021, 10, 686. https:// doi.org/10.3390/antiox10050686.

Nawaz AH, Amoah K, Leng QY, Zheng JH, Zhang WL and Zhang L (2021) Poultry Response to Heat Stress: Its Physiological, Metabolic, and Genetic Implications on Meat Production and Quality Including Strategies to Improve Broiler Production in a Warming World. Front. Vet. Sci. 8:699081

Nutritional manipulation to combat heat stress in poultry – A comprehensive review.Journal of Thermal Biology,98(2021)102915

Heat stress and poultry production: a comprehensive review.2023 Poultry Science 102:103141.

Line 68-69 “The diet compositions are shown in Table 1, and each nutritional level followed the breeder’s recommendation”. which kinds of diet, power  pellet should be displaied.

Table 1.not only listed AME and CP, the mainly nutrients should include Ca, NNP, Lys, Met+Cys, Thr, aleast.

Line 70-71 “Two birds that exhibited a similar BW to the average values of the pen were selected from each pen and euthanized by” at which day of age should be shown.

In Materials and Methods, usually the rectal temperature should be shown to display if the broilers is heat stress.

Line 93 this research studied FITC-d levels. However,why FITC should be determined has not reviewed in Introduction.

Line 104-105 “ approximately 5 g and with a similar surface area, was hung with a wire in a plastic case and stored at 4 °C for 5 days” usually,  stored at 4 °C for 1 day, not 5 days. Authors should double check how many meat, 5g? and how manys days ?And should supply the references.

Line 114-117 “Data are presented as the mean of six replicates (growth performance), twelve individual birds (blood parameters, gene expression, meat quality), or six birds (microbiota). Data were analyzed by using one-way (growth performance at the stater phase) or two-way (parameters excluding the left) analysis of variance (ANOVA)  ”Usually, if there is interation, one-way anova should be use to compare the difference between all the treatments.

Table 3, Usually, antioxidant enzymes such as SOD, CAT, GPx and glutathione reductase (GR) micromolecular antioxidants, such as glutathione (GSH) should be determined, however, SOD, CAT, GSH, not be determined.

The conclusion drew that “The HS-decreased muscle drip loss, lipid peroxidation, and glutathione content were also suppressed by BEPM supplementation”. however, the mainly effect should be listed with other figure or Table. Otherwise, based on Table 2-4 it is difficlut to drew the conclusion.

Author Response

We appreciate your valuable comments on the manuscript and answered the comments point-by-point as below. The title was changed to “Effect of feeding sugarcane bagasse-extracted polyphenolic mixture on the growth performance, meat quality, and oxidative and inflammatory status of chronic heat-stressed broiler chickens.” The bagasse-extracted polyphenolic mixture (BEPM) abbreviation was also changed to “SBPM," it was used throughout the responses. We appreciate your understanding.

 

1. Line 19-21 “Plasma lipid peroxidation content, inflammatory cytokines [interleukin (IL)-6, IL-β], corticosterone, and uric acid concentrations were significantly increased by HS, while the increases were attenuated by BEPM supplementation (P < 0.05)” in the Introduction, there is not enough review about inflammatory, and metabolism in blood, which can’t supply the evidence of study Plasma lipid peroxidation content, inflammatory cytokines. At least, the review should add enough information of stress on metabolism and inflammation in digestive organs.

• The above sentences are written in Abstract. Because of the word limitation, it is hard to add more information. Thank you for your understanding.

 

2. Line 35-38 “Broiler chickens raised under high-temperature conditions exhibit growth retardation and deterioration of their health, which is termed as heat stress (HS). Increased oxidative stress and inflammatory responses are the hallmarks observed in HS-treated chickens” the concept of heat stress isn’t right. Stress is the sum of defense mechanisms or non- specific responses of an organism when it is faced with abnormal situation or extreme demands (McEwen & Akil, 2020; Sahin et al., 2009). See Broiler production challenges in the tropics: A review/Vet Med Sci. 2021, 7:831–842. Heat stress cannot equal oxidative stress. Heat stress is one kind of very strong oxidative stress. In recent years, many published papers about heat stress of broilers.

• Thank you for your expert comments. We revised the related sentences (page 2, lines 49-55).

 

3. Line 68-69 “The diet compositions are shown in Table 1, and each nutritional level followed the breeder’s recommendation”. Which kinds of diet, powder pellet should be displayed.

• All the diets used in the investigation were made in mash form. The information was added to page 3, line 100.

 

4. Table 1. Not only listed AME and CP, but the mainly nutrients should also include Ca, NNP, Lys, Met+Cys, Thr, at least.

• Thank you for your expert comments. The information on the ingredients were added to Table 1.

 

5. Line 70-71 “Two birds that exhibited a similar BW to the average values of the pen were selected from each pen and euthanized by” at which day of age should be shown.

• The birds at 42 days of age were sacrificed. The information was added to page 2, line 101.

 

6. In Materials and Methods, usually the rectal temperature should be shown to display if the broilers is heat stress.

• Thank you for your expert comments. The body temperature was one of the results, the data was added to Results subsection 3.1 (page 5, lines 171-175) and the recording time/method also added to Method subsection 2.2 (page 2-3, lines 96-98).

 

7. Line 93; This research studied FITC-d levels. However, why FITC should be determined has not been reviewed in Introduction.

• FITC-d is one of the measurement methods, and the explanation may complicate the reader when mentioned in the Introduction section. The brief explanation was revised to make it easier for non-readers to understand (page 3, lines 108-110). Meanwhile, a description regarding the role of intestinal permeability in inflammation was added to the Introduction section, as it was not enough in the previous manuscript (page 2, lines 55-59).

 

8. Line 104-105 “ approximately 5 g and with a similar surface area, was hung with a wire in a plastic case and stored at 4 °C for 5 days” usually, stored at 4 °C for 1 day, not 5 days. Authors should double check how many meats, 5g? and how many days ?And should supply the references.

• We apologize for the mistakes in the cold storage period, which was done for 3 days correctly (page 4, line 137). Whilst muscle drip loss was measured for 1-day storage in many investigations, it takes a few days from the meat processed to reach the home table, including the transportation period. Therefore, we measured the drip loss after 3 days of storage. We think the storage period is dependent on the researcher’s concept. Thank you for your understanding.

 

9. Line 114-117 “Data are presented as the mean of six replicates (growth performance), twelve individual birds (blood parameters, gene expression, meat quality), or six birds (microbiota). Data were analyzed by using one-way (growth performance at the stater phase) or two-way (parameters excluding the left) analysis of variance (ANOVA) ”Usually, if there is interaction, one-way ANOVA should be use to compare the difference between all the treatments.

• Thank you for your expert comments. Our explanations regarding HS protocols and statistical analysis were not sufficient. The chicks at the starter phase were not subjected to the HS treatment due to the equipment limitation (upper-temperature setting). Therefore, the HS treatment began at 11 days of age (see Subsection 2.1). Thus, it is difficult to analyze the growth performance data at the starter phase (0-10 d) by two-way ANOVA because of one temperature. Thank you for your understanding.

 

10. Table 3, Usually, antioxidant enzymes such as SOD, CAT, GPx and glutathione reductase (GR) micromolecular antioxidants, such as glutathione (GSH) should be determined, however, SOD, CAT, GSH, not be determined.

• Thank you for your expert comments. The study was a preliminary trial and did not aim to clarify the detailed mechanism of suppressions of inflammation and oxidative damage due to SBPM supplementation. The description to the point is stated more clearly, as it was not enough in the previous manuscript (page 8, lines 317-319). The issues will be addressed in future investigations.

 

11. The conclusion drew that “The HS-decreased muscle drip loss, lipid peroxidation, and glutathione content were also suppressed by BEPM supplementation”. However, the mainly effect should be listed with another figure or Table. Otherwise, based on Table 2-4 it is difficult to drew the conclusion.

• The sentence indicated above was written in Abstract, and it was not a conclusion but an explanation of the results.

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the manuscript identified as Animals- 3189446 and concerns the evaluation of the effects of bagasse-extracted polyphenolic mixture (BEPM) supplementation on the harmful effects of chronic heat stress (HS) in broiler chickens. Manuscript is well written and contains ample experimental data, which along with the relevant literature reports have been properly presented and discussed. 

Thus, I recommend its publication provided that authors will improve the following:

Major

Although main objective of the manuscript is the broiler chickens feeding with polyphenolic mixtures of squeezed sugarcane bagasse, authors did not include any info concerning its origin (who provided the material, what was its condition it was dried, under what conditions etc). In addition, the analytical data concerning its polyphenolic content and possible antioxidant properties are missing (eg. total phenolic content, assessment of individual polyphenols presence, antioxidant capacity determination etc). Since it is well established that the polyphenolic content of this material depends greatly on their production (Line 46, refs 10,11), treatment etc authors have to provide all these data in order the respective feeding data become useful.

Authors have to include these data and discuss them in respect their results (abstract, discussion, conclusions parts)

Minor

A.    Table 1: materials characterized in footnote as Vitamin/minerals do not fall into these categories (eg. tocopherol acetate, 2-methyl-1,4-napthoquinone sodium bisulfite, thiamine nitrate etc).

Also, in crude protein heading there is no footnote for 2.

B.    Tables 2, 3, 4 there are no footnotes for a, b, c, d etc 

Author Response

We appreciate your valuable comments on the manuscript and answered the comments point-by-point as below. The title was changed to “Effect of feeding sugarcane bagasse-extracted polyphenolic mixture on the growth performance, meat quality, and oxidative and inflammatory status of chronic heat-stressed broiler chickens.” The bagasse-extracted polyphenolic mixture (BEPM) abbreviation was also changed to “SBPM," it was used throughout the responses. We appreciate your understanding.

 

1. Although the main objective of the manuscript is the broiler chickens feeding with polyphenolic mixtures of squeezed sugarcane bagasse, authors did not include any info concerning its origin (who provided the material, what was its condition it was dried, under what conditions etc). In addition, the analytical data concerning its polyphenolic content and possible antioxidant properties are missing (eg. total phenolic content, assessment of individual polyphenols presence, antioxidant capacity determination etc). Since it is well established that the polyphenolic content of this material depends greatly on their production (Line 46, refs 10,11), treatment etc. Authors have to provide all these data in order the respective feeding data become useful. Authors have to include these data and discuss them in respect of their results (abstract, discussion, conclusions parts).

Thank you for your expert comments. The following information was added in a newly generated Subsection 2.1. Preparation of BEPM; “SB was treated with alkali solution (1% sodium hydroxide solution for 2 hours), and the soluble fraction was adjusted pH under 4.0 and precipitated by 35% hydrochloric acid. The insoluble fraction obtained was made dry and powdered. BEPM consists of 5-10% polyphenol, <5% moisture, 50-70% diatom earth, and other constituents. The oxygen radical absorbance capacity (ORAC) value was more than 10 μmoles per g as a Trolox equivalent.” The detailed polyphenol compositions were not identified because of technical limitations.  

 

2. Table 1: materials characterized in footnote as Vitamin/minerals do not fall into these categories (eg. tocopherol acetate, 2-methyl-1,4-napthoquinone sodium bisulfite, thiamine nitrate etc). Also, in crude protein heading there is no footnote for 2.

• The footnote in Table 1 is the components of vitamin/mineral premix. And, we are sorry that we could not understand what the latter query meant.

 

3. Tables 2, 3, 4 there are no footnotes for a, b, c, d etc 

• The explanation for the superscript letters was mentioned in Table 2 and 3. We apologize for missing the information in Table 4, and the information was added to the footnote.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you very much for entrusting me with this article for review.

After reading the content of this text, I present my comments and recommendations:

1. Title: this version does not sound good because it does not reflect the essence of the research. I think it should start with the word "impact...".

2. Introduction: a very short fragment, which, in my opinion, should be expanded, at least in the fragment concerning bagasse.

3. Materials and methodology:
- line 58: here, there should be an exact indication of the age of these chickens - "1 day-old" (not "day-old").
- lines 105-106: I recommend including the formula for calculating exudate (there is only a verbal description).
- I missed information about the origin of bagasse and the method of obtaining it, which is essential information for the reader. Without primary data, the experiment cannot be repeated.
- For statistical analyses, please specify what analyses were performed. In lines 112-119, there was no information about conducting a correlation analysis, while in the "Discussion" fragment (lines 211-212), there was information about the positive correlation found.

4. The article lacks formulated conclusions. I believe that they should be separated into separate chapters.

5. The article lacks information on bagasse and its properties (e.g., polyphenol content discussed in the article in the appropriate place). If the Authors characterized these properties in their previous publications, they should refer to them. I noticed that the primary author (corresponding author) cited his publications, numbering 5 (publications no. 3, 16, 17, 19, and 37), which is significant considering the total 41 publications.

6. Table 1: I believe that the expression "bird age" should be added next to the word "ingredients" in the first row of the table.

7. Table 3: In the data for "IL-1B," there are missing symbols regarding the homogeneity of results, and they should be supplemented. It is similar to Table 4, where these symbols are missing in the "Drip loss" results.

Author Response

1. Title: this version does not sound good because it does not reflect the essence of the research. I think it should start with the word "impact...".

• Thank you for your advice. We revised the title to fit the content as follows: “Effect of feeding sugarcane bagasse-extracted polyphenolic mixture on the growth performance, meat quality, and oxidative and inflammatory status of chronic heat-stressed broiler chickens.” The abbreviation of the bagasse-extracted polyphenolic mixture (BEPM) was also changed to SBPM, and it was used throughout the responses.

 

2. Introduction: a very short fragment, which, in my opinion, should be expanded, at least in the fragment concerning bagasse.

• Reviewer 1 suggested revising the definition of heat stress, and the section was therefore expanded. Several reports investigate bagasse; however, they focus on bioprocessing, materials, and functional residue in composting, each of which is far from the aim of the present investigation. Therefore, the information was not shown in the Introduction section for readability. Thank you for your understanding.

 

3. Materials and methodology, - line 58: here, there should be an exact indication of the age of these chickens - "1 day-old" (not "day-old").

• We used 0 day-old chicks in the manuscript. However, in our previous publication, a few reviewers indicated that “0 day-old” sounds strange and should be shown as “day-old” when neonatal chicks were used. Therefore, this style was also used in the present manuscript. To this point, we await the Editor’s opinion/judgement. Thank you for your understanding.

 

4. Lines 105-106: I recommend including the formula for calculating exudate (there is only a verbal description).

• The following formula was added (pages 4, line 140): Drip loss (%) = [(initial weight - the weight after cold storage) / initial weight] × 100.

 

5. I missed information about the origin of bagasse and the method of obtaining it, which is essential information for the reader. Without primary data, the experiment cannot be repeated.

The article lacks information on bagasse and its properties (e.g., polyphenol content discussed in the article in the appropriate place). If the Authors characterized these properties in their previous publications, they should refer to them. I noticed that the primary author (corresponding author) cited his publications, numbering 5 (publications no. 3, 16, 17, 19, and 37), which is significant considering the total 41 publications.

• Thank you for your comments. Reviewer #2 also commented on a similar point, and brief manufacturing information on SBPM was added in Subsection 1. More detailed information cannot be disclosed because of the trade secret in manufacturing. Thank you for your understanding.

 

6. For statistical analyses, please specify what analyses were performed. In lines 112-119, there was no information about conducting a correlation analysis, while in the "Discussion" fragment (lines 211-212), there was information about the positive correlation found.

• The methods for statistical analyses were shown in Subsection 2.5. We apologize for missing the method using correlation analysis in meat quality parameters. The following information was added to page 4, lines 153-154; “Pearson’s coefficient analysis was used to analyze the correlation in meat quality parameters.”

 

7. The article lacks formulated conclusions. I believe that they should be separated into separate chapters.

• We apologize for lacking the Conclusion section. It was added as Subsection 5.

 

8. Table 1: I believe that the expression "bird age" should be added next to the word "ingredients" in the first row of the table.

• Thank you for your advice. The word was added.

 

9. Table 3: In the data for "IL-1B," there are missing symbols regarding the homogeneity of results, and they should be supplemented. It is similar to Table 4, where these symbols are missing in the "Drip loss" results.

• Thank you for the comments. In our understanding, it is generally not necessary to put the superscript letters identifying the statistical difference once the interaction effect with Temp. × Diet was not observed. Thank you for your understanding and consideration.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I have no additional comments.