Review Reports

S.No

Reviewer 01: Comments

Responses

1.       

The abstract section is relatively long, please try to shorten it and focus on key quantitative results/insights.

Thanks for the suggestion.

The abstract has been revised as suggested please.

2.       

Improve the introduction and provide sufficient background and include all relevant references

Thanks for the suggestion.

The introduction is improved with more relevant background and references please.

3.       

Please italicize all species names. Please go through the text and adapt.

Thank you for the suggestion. Completed, we went through the text and followed the suggestions (highlighted)

4.       

Please be consistent with the use of numbers and units. Sometimes a space is used between number and unit, sometimes not (even for the same parameter). Please go through the text and adapt for consistency.

Thanks for the suggestion. Gone through and corrected please.

5.       

Lines 51-53: please provide a reference.

Done as suggested,

·         Please refer to the line 50-52 highlighted as red color.

6.       

Please mention all abbreviations in full once they are used in the text. This is currently not the case (for example PFA).

Done as suggested please (line 58).

7.       

Lines 79-81: please provide a reference.

Done as commented,

·         Please refer to the lines 79-81 highlighted as red color.

8.       

The authors should place greater emphasis on the novelty of this research. What is the novelty of this research? What has already been done in the literature? Has HA already been tested as an additive in animal feed? Research gaps should be identified and novelty should be explicitly stated.

Thanks for thoughtful comment. We have added the following novelty statements please.

Maize plays a central role in poultry feed formulation, and the abundant HA present in its tissues serves as a novel phytogenic feed additive. However, the utilization of the HA extracted from the roots of maize are rarely investigated. This study extracted HA from different maize varieties to analyze its concentration and its impact on in vitro antibacterial activity against pathogenic E. coli, as well as its in vivo effects on broiler performance, carcass yield, and serum parameters, Please refer to the lines 77-86 highlighted as red color.

9.       

Please provide more details regarding the sonication device (amplitude, power, frequency, etc).

Thanks for the suggestion. Done,

Samples were sonicated at frequency 40kHz, power 200W and amplitude 3mm using an ultrasonic cleaner for 10 min.

·         Please refer to the lines 104-105 highlighted.

10.   

Line 154 & Line 161: remove 0 in “06”.

Thank you for the suggestion. Done as requested.

·         Please refer to lines 156&162 (highlighted) 

11.   

Please mind significant figures in Table 2.

We thank the reviewer for pointing out the issue with significant figures in Table 2. The table has been revised for consistency and appropriate precision. Ingredient compositions are uniformly presented with two decimal places, while nutritional values are rounded to suitable figures, with energy shown without decimals and nutrients standardized to two decimals. The revised table now presents the data more clearly and consistently. Please refer to Table 2.

·         Please refer to lines 166-167 (highlighted) 

12.   

The authors use many abbreviations, an abbreviation list might facilitate reading.

List updated.

·         Please refer to lines 431-433 (highlighted)

13.   

Table 3, mention in the accompanying text that the HA was determined in the seedling roots. In this case this column can be removed from the table.

Column removed as suggested please.

·         Please refer to Table 3- lines 215-217 (highlighted) 

14.   

The authors focused on the hydroxamic acids present in the seedling roots. However, what is the concentration in the kernels? Is the concentration in the roots significant compared to that in the kernels fed to the broilers?

·         Thanks for thoughtful comment. Actually, Hydroxamic acids (Benzoxazinoids (BXs)) are not present in kernels. They are natural defense compounds which protect plants from pathogens and insect during early days of the seedling life. The BX biosynthetic pathway remain dormant in seeds and is activated with germination of seeds. We have quantified hydroxamic acid from different part of plants including roots, stem, leaves and seeds we found that the root contains highest concentration of hydroxamic acid. And we found no hydroxamic acid in seeds.

15.   

Please introduce scale bars in the Figures presented.

Introduced please find in Figures 4-6.

16.   

Line 224-225: it seems that this text has another formatting compared to the rest of the Figure’s caption.

Format corrected.

·         Please refer to lines 224-228 (highlighted)

17.   

Figure 4 is very difficult to read because it is too small. Please reorganize and increase its size.

Figure 4 is re-structured for better understanding.

·         Please refer to lines 256-257. 

18.   

Also consider improving the quality of Figure 5 to facilitate reading.

Thank you so much. The figure 5 quality has been improved.

19.   

Line 276: “Hemoglobin” does not require a capital letter.

Thank you for improving our manuscript. Done as suggested.

·         Please refer to line 288 (highlighted) 

20.   

Lines 288-290: please provide a reference.

The reference has been added.

·         Please refer to line 309 (highlighted) 

21.   

Line 302: there is a period too much.

Corrected,

·         Please refer to lines 321 (highlighted) 

22.   

The conclusion section is very short. Please include some key (quantitative) results and insights.

Thanks for the suggestion. Conclusion is revised as suggested.

·         Please refer to lines 385-395 (highlighted) 

23.   

The authors prepared extracts from the maize seedling roots and measured HA content via HPLC. However, how sure can the authors be that the observed effects are related (solely) to HA? There will be many more components present after extraction most probably?

Thanks for thoughtful comment. We have maize ­HA mutant lines (maize that cannot produce HA). The extract from HA mutant lines was used as negative control. The extract from roots of these lines possess all component except HA. These mutant lines were used by one of the coauthor in his their previous study (Benzoxazinoid Metabolites Regulate Innate Immunity against Aphids and Fungi in Maize (Plant Physiology, Oxford Academic). Therefore we confirmed that the antibacterial effects are because of HA not because of other components in the root extract.

24.   

The authors mention the measured hydroxamic acids, but which ones specifically? Which standards were used of which hydroxamic acid to determine the concentration? Please specify. Would it be possible to present a chromatogram in the supplementary information of a typical experiment?

·         Thanks for thoughtful comment. The reviewer is right there are many hydroxamic acids (Benzoxazinoids (BXs)) in maize, the most abundant among them is  2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). We have used DIMBOA to measure BX concentration in the maize roots extract. For this purpose, DIMBOA standard was used to quantify the concentration of hydroxamic acid in different maize verities. Chromatogram from the BX standard (DIMBOA) and maize verity is presented as supplementary information (Figure S2).

·         Please refer to the lines 116-118 highlighted.

S.No

Reviewer 02: Comments

Responses

1.       

Improve the introduction and provide sufficient background and include all relevant references

Thanks for the suggestion.

The introduction is improved with more relevant background and references please.

2.       

in line 36-37, It is recommended to provide detailed information on the dosage concentration, and do not use abbreviations for the dosage method, such as “mg/kg”.

Thank you for the suggestions. We have revised the text as per suggestion.

·         Please refer to lines 27-30 (highlighted) 

3.       

Revise minor grammatical issues in the manuscript, such as change “ml” to “mL”, “hrs” to “h”.

Revised please (highlighted) 

4.       

Table 3 shows the HA content in different types of varieties of Maize being tested. What is the extraction rate in this experiment? What is the purity of HA used presently?

Thanks for the comments. The extraction rate of HA in this experiment is µg/g of roots.

Whereas based on the retention time of HPLC analysis we found that the HA is 100% pure

5.       

It is suggested to enhance the clarity of the figures.

Thank you for the suggestion. We have improved the quality of figures.

6.       

It is suggested to clearly indicate MIC in a table format.

MIC is presented in Table S2 (Supplementary) as suggested please.

7.       

The author has analyzed the growth-promoting mechanism of HA. Then, what is its antibacterial mechanism?

Thank you for the suggestion. Maize-derived HA may provide several mechanisms of action, including direct inhibition of gut pathogens, chelation of excess metals, and modulation of host antioxidant defenses [19].

A key feature of HA is their strong iron-chelating ability, which bacteria normally exploit via siderophores to acquire essential iron [18].

·         Please refer to lines 79-81 (highlighted) 

8.       

It is suggested that the author enrich the content of the conclusion.

Thanks for the suggestion. Conclusion is revised as suggested.

Please refer to lines 385-395 (highlighted) 

9.       

Finally, it is suggested that the author enrich the content of the illustrations so that readers can quickly obtain the results of the work.

The illustrations content is revised and enriched as suggested. Please refer to the captions of all figures (highlighted)