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  • Xiaojie Ren 1,2,†,
  • Yan Zhang 1,2,† and
  • Weiren Yang 1,*
  • et al.

Reviewer 1: Tossaporn Incharoen Reviewer 2: Ion Brinza Reviewer 3: Sajid Khan Tahir Reviewer 4: Tarique Hussain

Round 1

Reviewer 1 Report

Overall content of this manuscript is good including interesting parameters to explain the beneficial effects of probiotic. However, few additions would help to provide more clarity.

1. Scientific name should be in italic (line 37, 210, 242, 389, 487, 490, 505, 506, and 508).

2.     Layers or broilers? (line 57).

3.     The AA broiler management guideline is required to be added as a reference (line 62).

4.     Was the nutrient calculation base on NRC? Or other, line 75 needs a Ref.

5.     ME vale was wrong. Please recheck an energy unit. Should it be MJ/kg? (Table 1)

6.     What is the gender of those broilers? Was it a mixed-sex broiler? If so, the mixed-sex bird was not suitable for performance determination because they are different growth rates. Anyway, is there management to be an equal number of each sex within each replication? If so, you should mention the procedure in the manuscript (line 58-59).

7.     How old were those broilers began the experiment? Initial weight = 42±0.5 g, it must be one-day-old. As authors described “one-week-old”, it must be revised (line 58-59).

8.     Intestinal tissue collected from fasting bird could not be accepted for analyzing intestinal morphology. If so, some citations should be added (line 81-82).

9.     Adding citation into method of Serum Biochemistry should be done (line 86-88).

10.  “jejunum” should be changed to “jejunal” to be as adj (line 97 and 140).

11.  Is “DAG” must be “ADG”? (line 152)

12.  Author have to check the sentence in line 166-167, 178-179, 195-196, 206-207 and 218-219 because there is no row of table, these results showed as bar.

13.  What is a meaning of “Velvet concealed ratio”? Please explain how to measure this paramenter.

14.  Bacillus subtilis BC02 could improve growth performance of starter broiler, if so author should rewrite the conclusion.

English quality of manuscript is satisfied. 

Author Response

Response to Reviewer 1 Comments

 

Thank you very much for your thoughtful and useful comments and suggestions. Those comments are all valuable and very helpful for revising and improving our manuscript. The parts that reviewers referred have been noted by using red type in the revised manuscript text. We have included almost all of your suggestions and below we present a point-by-point response to your comments. 

 

Point 1: Scientific name should be in italic (line 37, 210, 242, 389, 487, 490, 505, 506, and 508).

 

Response 1: We are really sorry for our careless mistakes. Thank you very much for your careful checks. We have corrected and re-checked the other words of the revised manuscript (Line 39, 213, 246, 395, 495, 498, 520, 521 and 523).

Point 2:  Layers or broilers? (line 57).

Response 2: Thank you very much for your excellent comment and suggestion. We're sure it's broilers. The purposes of this study were to investigate the effects of Bacillus subtilis BC02 supplementation on broiler performance, antioxidant capacity and cecal microbes.

Point 3:  The AA broiler management guideline is required to be added as a reference (line 62).

Response 3: Thank you very much for your excellent comment and suggestion. The reference has been added at line 70 and 509.

Point 4:  Was the nutrient calculation base on NRC? Or other, line 75 needs a Ref.

Response 4: Thank you very much for your excellent comment and suggestion. In the present study, the diets were formulated according to Feeding Standard of Chicken of the People’s Republic of China, NY/T 33-2004, which has been added at line 64-65.

Point 5:  ME vale was wrong. Please recheck an energy unit. Should it be MJ/kg? (Table 1)

Response 5: We are really sorry for our careless mistakes. Thank you very much for your careful checks. Table 1 “ME, kcal/kg” was changed to “ME, MJ/kg”.

Point 6:  What is the gender of those broilers? Was it a mixed-sex broiler? If so, the mixed-sex bird was not suitable for performance determination because they are different growth rates. Anyway, is there management to be an equal number of each sex within each replication? If so, you should mention the procedure in the manuscript (line 58-59).

Response 6: Thank you very much for your excellent comment and suggestion. In the present study, all broilers used in trial are male. This detail have been added in the parper. （Line 59）“(Arbor Acres, 42±0.5 g) broilers” was changed to “(Arbor Acres, 42±0.5 g) male broilers”.

Point 7:  How old were those broilers began the experiment? Initial weight = 42±0.5 g, it must be one-day-old. As authors described “one-week-old”, it must be revised (line 58-59).

Response 7: We are really sorry for our careless mistakes. Thank you very much for your careful checks. (Line 59) “one-week-old” was changed to “one-day-old”.

Point 8:  Intestinal tissue collected from fasting bird could not be accepted for analyzing intestinal morphology. If so, some citations should be added (line 81-82).

Response 8: Thank you very much for your excellent comment and suggestion. The sampling procedure adopted in this trial was referred antoher scholars’ study (Line84-85, 510-512).

Reference:

Liu, Y.; Wang, Q.; Liu, H.; Niu, J.; Jiao, N.; Huang, L.; Jiang, S.; Guan, Q.; Yang, W.; Li, Y. Effects of dietary Bopu powder supplementation on intestinal development and microbiota in broiler chickens. Front Microbiol. 2022, 13, 1019130.

Point 9: Adding citation into method of Serum Biochemistry should be done (line 86-88).

Response 9: Thank you very much for your excellent comment and suggestion. “(Roche Diagnostic System Inc., USA).” was changed to ”(Roche Diagnostic System Inc., USA) according to the methods described in Liu et al. (2022) [25]” (Line 91-92, 513-515).

Reference:

Liu, Y.; Li, Y.; Niu, J.; Liu, H.; Jiao, N.; Huang, L.; Jiang, S.; Yan, L.; Yang, W. Effects of dietary Macleaya cordata extract containing isoquinoline alkaloids supplementation as an alternative to antibiotics in the diets on growth performance and liver health of broiler chickens. Front Vet Sci. 2022, 9, 950174.

Point 10: “jejunum” should be changed to “jejunal” to be as adj (line 97 and 140).

Response 10: We are really sorry for our careless mistakes. Thank you very much for your careful checks. (Line 97 and 145) “jejunum” was changed to “jejunal”.

Point 11: Is “DAG” must be “ADG”? (line 152)

Response 11: Thank you very much for your excellent comment and suggestion. In the present study, the meaning of “ADG” is “average daily gain”. We haven’t use “DAG” to analysis the growth performance, which may be improved in the further study.

Point 12: Author have to check the sentence in line 166-167, 178-179, 195-196, 206-207 and 218-219 because there is no row of table, these results showed as bar.

Response 12: Thank you very much for your excellent comment and suggestion. We have check it and make it be approriate. In this study, several results were exhabit in forms of figure so that to reflect the results and tendance directly (Line 164-165, 175-176, 189-190, 204-205, 215-216, 232-233, 241-242, 256-257, 282-283, 295-296, 313-314, and 334-335).

Point 13: What is a meaning of “Velvet concealed ratio”? Please explain how to measure this paramenter.

Response 13: Thank you very much for your excellent comment and suggestion. The “velvet concealed ratio” is the ratio of villi height to the crypt depth. In short, the villi height was measured from the villi tip to the villi base, and the crypt depth was measured from the intervillous valley to the basement membrane.

Point 14: Bacillus subtilis BC02 could improve growth performance of starter broiler, if so author should rewrite the conclusion.

Response 14: Thank you very much for your excellent comment and suggestion. Although our results indicated that dietary supplementation of Bacillus subtilis BC02 improved growth performance of starter broilers, while this supplementation also improved the 0-28 d and 0-42 d F/G of broilers. Therefore, our conclusions summarized that dietary supplementation of Bacillus subtilis BC02 can improve growth performance of broiers (Line 150-154). 

Please see the attachment.

We hope the revised manuscript will meet the requirements. We sincerely appreciate your warm work again.

Author Response File: Author Response.docx

Reviewer 2 Report

In this article, the authors investigated the effects of Bacillus subtilis BC02 supplementation on broiler performance, antioxidant capacity and cecal microbes. The data of the study reveals that dietary Bacillus subtilis BC02 supplementation can improve the growth performance, antioxidant capacity and intestinal flora structure in broilers.

 

The work is of interest both for meat bird breeders and for researchers in the field in order to increase the growth capacity and the health status of birds dedicated to meat production.

The introduction provides enough details to understand the importance of the study as well as the theme, its general notions. The materials and methods are clearly described and can be reproduced. The results are presented coherently and clearly. The conclusions are supported by the results.

 

Minor corrections:

row 26 - the space between the group and the point must be removed

Material and methods. Row 58-59 broilers were randomly divided into 3 treatments of 6 replicates. The details of the treatments and the replicates are suggested. It is suggested to do an experimental design for a better understanding of the study.

It is suggested to write genres in italics. Ex. row 37 Bacillus subtilis; row 301 Barnesiellaceae.

Author Response

Response to Reviewer 2 Comments

 

Thank you very much for your thoughtful and useful comments and suggestions. Those comments are all valuable and very helpful for revising and improving our manuscript. The parts that reviewers referred have been noted by using red type in the revised manuscript text. We have included almost all of your suggestions and below we present a point-by-point response to your comments. 

 

Point 1: Row 26 - the space between the group and the point must be removed.

 

Response 1: We are really sorry for our careless mistakes. Thank you very much for your careful checks. According to your suggestion, we have removed the space between the group and the point (Line 28).

 

Point 2: Material and methods. Row 58-59 broilers were randomly divided into 3 treatments of 6 replicates. The details of the treatments and the replicates are suggested. It is suggested to do an experimental design for a better understanding of the study.

 

Response 2: Thank you very much for your excellent comment and suggestion. According to your suggestion, we have rephrased the experimental design and layers management part (Line 60-71) for a better understanding of the revised manuscript.

 

Point 3: It is suggested to write genres in italics. Ex. row 37 Bacillus subtilis; row 301 Barnesiellaceae.

 

Response 3: Thank you very much for your excellent comment and suggestion. According to your suggestion, We have corrected and re-checked the other words of the revised manuscript (Line 39, 213, 246, 395, 495, 498, 520, 521 and 523) and changed “Lactobacillales, lactobacillaceae, Barnesiellaceae, lactobacillus_aviarius, Bacteroides_caecigallinarum, Campylobacterales, Campylobacteria, Barnesiellaceae, Lactobacillaceae,   Lactobacillales, Campylobacterales and Campylobacteria” to “Lactobacillales, lactobacillaceae, Barnesiellaceae, lactobacillus_aviarius, Bacteroides_caecigallinarum, Campylobacterales, Campylobacteria, Barnesiellaceae, Lactobacillaceae,   Lactobacillales, Campylobacterales and Campylobacteria” (Line 305-312).

Please see the attachment.

We hope the revised manuscript will meet the requirements. We sincerely appreciate your warm work again.

Author Response File: Author Response.docx

Reviewer 3 Report

The present study is aimed to investigate the effects of supplementation of Bacillus subtilis on the production performance, redox status, and cecal microbes in broilers. The study is well-designed and data collection is appropriate with expert statistical analyses. Few section-wise suggestions/changes are required to improve the manuscript further;

Abstract:

1. Remove the abbreviations like ADFI, F/G, SOD, GSH, and MDA, as they are not used again in the abstract.

2. In line 15, replace "A total of 288 one day Arbor Acres..." with "A total of 288 one day old Arbor Acres".

2. In line 16, replace "were" with "was" and "basal diets" with "basal diet".

3. Line 19, "Meanwhile, BS500......". please break the lengthy statement into two or three.

4. Line 27-28, what is meant by "intestinal flora structure".

Introduction:

1. In line 36, replace "more and more attention" with "more attention".

2. In line 42, replace "Can improve growth performance" with "Can improve the growth performance".

3. In line 54, remove the statement at the end of the objectives "and to provide a application of......

Materials and Methods:

1. What is the basis for selecting doses @ 250 and 500? The reason for the selection of dose quantity could be mentioned.

2. In line 61, replace "is" by "was".

3. In line 62, replace "are" by "were".

4. In lines 66-67, rephrase the statement, "The control group were fed basal diets, and treatment groups were" as " The control group was fed basal diet and the treatment groups were ".

5. In lines 69-70, rephrase the statement, ", which the endospore content..." as ", with endospore content..."  

6. In lines 80-81, rephrase the statements, The jejunum was separated under..... and "The jejunum was stored..." as "After slaughtering, the jejunum was separated, washed, and stored at -80 ºC for mRNA analysis.

7. In line 84, add "for cecal microbial analysis" at the end of the sentence as "stored at -80 ºC for cecal microbes analysis".

8. In line 103, rephrase the sentence "Use the kit to extract...." by correcting the grammar.

9. In lines 114-117, please rephrase the long sentence "The microbial DNA.... as "The microbial DNA from the cecal contents was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Biotek, USA). Further, the extracted DNA concentration and purity were analyzed by TBS-380 and NanoDrop2000 respectively".

10. In line 124, please add "was performed" as "On-machine sequencing was performed using....".

11. In line 125, replace "are" with "were".

12. In line 135, replace "tests" with "test".

13. In line 136, replace "are" with "were".

14. In line 138, rephrase "Simpson were analyzed..." as "Simpson were analyzed by one-way ANOVA".  

Results

1. Line 148, please remove the statement "But there were no difference in ADG,...." because the authors already compared the growth effects of BS with the control. Further, it can be elaborated when there is the difference among the treatment levels, no need to write the similar effects.

2. In line 149, please crosscheck the statement with the results given in Table 3. I think the F/G ratio is significantly different at 0-28d and 0-42d. Also, please correct it in the discussion.

3. In Table 3, please add a proper abbreviation to "1d, g, 14d,g, 28d,g, 42d,g" as 1d-LBW,g, etc so that the reader can easily get it as live body weight.

4. Line 159, please follow the comments made in S. No.1 of the results section.

5. In line 160, please crosscheck the abbreviation for total cholesterol as here it is "TCHO" while in materials and methods, it is "TC".      

6. In lines 171-172 and 200, please follow the comments made in S. No.1 of the results section.

7. In line 211, please check the result of mRNA expression IL-6 as it is written as significant once and then non-significant in the last sentence.  

Discussion:

1. In lines 346-347, replace "positive relation" with "positively related".

2. In lines 356-357, replace "can increase nutrient absorption" with "can increase the nutrient absorption".

3. In lines 360-361, replace "resulted in an increase in the abundance of Bacteriodetes cecal,...." as "resulted in an abundance of cecal Bacteriodetes, improved nutrient absorption, energy, and fat metabolism which ultimately enhanced the growth performance."

4. In line 416, replace "the previous study which" with "the previous studies in which".

5. In line 419, replace "may" with "may be".

6. In line 420, replace "also was" with "was also".

Conclusion:

 

Please rephrase the conclusion carefully. What is meant by "structure of intestinal flora"? I am unable to find any analysis in the manuscript that measured the structure of intestinal flora. 

Added already in comments and suggestions for authors sections.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

The article by Ren et al., 2023 titled “Effects of Dietary Bacillus subtilis BC02 Supplementation on 2 Growth Performance, Antioxidant Capacity and Cecal Mi-3 crobes in Broilers” was carefully reviewed and suggested the following changes.

1.       There are several studies conducted on this aspect, what is the novel idea in this one?

2.       The desired concentration was used 250 and 500 mg/kg Bacillus subtilis BC02. Was this concentration optimized or referred by previous studies?

3.       What is the dose range of probiotic bacteria, one previous study indicate 2 × 109 CFU/g.

4.       Bate diversity; the correct spelling should be beta diversity?

5.       Please make figure 11B prominent.

6.       First sentence of discussion needs to be rephrased.

7.        There are some minor mistakes of English, needs to be corrected.

There are minor English errors, need to be corrected.  

Author Response

Response to Reviewer 4 Comments

 

Thank you very much for your thoughtful and useful comments and suggestions. Those comments are all valuable and very helpful for revising and improving our manuscript. The parts that reviewers referred have been noted by using red type in the revised manuscript text. We have included almost all of your suggestions and below we present a point-by-point response to your comments. 

 

Point 1: There are several studies conducted on this aspect, what is the novel idea in this one?

 

Response 1: Thank you very much for your excellent comment and suggestion. At present, some studies have reported that Bacillus subtilis plays a positive role in animal growth, diseases prevention and intestine health of broilers. However, the growth rates and reproductive cycle of Bacillus subtilis in the intestinal tract are inconsistent, which may limit them to perform better. Meanwhile, the Bacillus subtilis BC02 used in the test was isolated and bred from the intestinal tract of healthy animals, which ensured its growth rate and reproductive cycle. On this basis, this study was conducted to explore its beneficial effects on growth performance, antioxidant capacity, and cecal microbes of broilers. Truly, the comparisons among Bacillus subtilis BC02 and other Bacillus subtilis which has been reported are required to be conducted in the future.

 

Point 2: The desired concentration was used 250 and 500 mg/kg Bacillus subtilis BC02. Was this concentration optimized or referred by previous studies?

 

Response 2: Thank you very much for your excellent comment and suggestion. The concentrations used in animals trials were referred to other scholars’ reports. In our study, the Bacillus subtilis BC02 endospore content is 4.0×109 cfu/g, and we set two concentrations 250 an 500 mg/kg (endospores content is 1×109 and 2×109 cfu/kg feed), which was consistent with Gao et al. (2017). Additionally, Wang et al. (2022) also investigated the effects of dietary supplementation of 1×109 cfu/kg Bacillus subtilis KC1 on growth performance, immune response, heat stress tolerance, and disease resistance of broilers.

Reference: 

  • Gao, Z.; Wu, H.; Shi, L.; Zhang, X.; Sheng, R.; Yin, F; Gooneratne, R. Study of Bacillus subtilison growth performance, nutrition metabolism and intestinal microflora of 1 to 42 d broiler chickens. Anim Nutr. 2017, 109-113.
  • Wang, J.; Ishfaq, M.; Miao, Y.; Liu, Z.; Hao, M.; Wang, C.; Wang, J.; Chen, X. Dietary administration of Bacillus subtilisKC1 improves growth performance, immune response, heat stress tolerance, and disease resistance of broiler chickens. Poult Sci. 2022, 101, 101693.

 

Point 3: What is the dose range of probiotic bacteria, one previous study indicate 2 × 109 CFU/g.

 

Response 3: Thank you very much for your excellent comment and suggestion. At present, plenty of studies have indicated that various supplementation doses of probiotic bacteria are beneficial for growth performance, immunity, antioxidant capacity, meat quality, and intestinal barrier of broiler chickens in different degrees. Summarized from the present reports have been published, except the 1×109 and 2×109 cfu/kg of feed used in this study, several scholars also chosed the 5×108 cfu/kg (Rhayat et al., 2017), 2×1010 cfu/kg, 3×1010 cfu/kg (Bai et al., 2016), and 1×1011 cfu/kg (Mohamed et al., 2022) of feed to investigate its effects.

Reference:

[3] Rhayat, L.; Jacquier, V.; Brinch, K.S.; Nielsen, P.; Nelson, A.; Geraert, P.A.; Devillard, E. Bacillus subtilis strain specificity affects performance improvement in broilers. Poult Sci. 2017, 96, 2274-2280. [4] Bai, K.; Huang, Q.; Zhang, J.; He, J.; Zhang, L.; Wang, T. Supplemental effects of probiotic Bacillus subtilis fmbJ on growth performance, antioxidant capacity, and meat quality of broiler chickens. Poult Sci. 2017, 96, 74-82.

[5] Mohamed, T.M.; Sun, W.; Bumbie, G.Z.; Dosoky, W.M.; Rao, Z.; Hu, P.; Wu, L.; Tang, Z. Effect of dietary supplementation of Bacillus subtilis on growth performance, organ weight, digestive enzyme activities, and serum biochemical indices in broiler. Animals (Basel). 2022, 12, 1558.

 

Point 4:  Bate diversity; the correct spelling should be beta diversity?

 

Response 4: We are really sorry for our careless mistakes. Thank you very much for your reminder. According to your suggestion, we have corrected it and re-checked the other words spelling errors in the revised manuscript (Line 247-248, Line 258, Line 417).

 

Point 5:  Please make figure 11B prominent.

 

Response 5: Thank you very much for your excellent comment and suggestion. According to your suggestion, we have re-edited figure 11 and added the description of figure 11B in the revised manuscript (Line 309-311, Line 313-314).

 

Point 6: First sentence of discussion needs to be rephrased. 

 

Response 6: Thank you very much for your excellent comment and suggestion. First sentence of discussion ”With the deepening of probiotic research, it has been gradually recognized that dietary Bacillus subtilis supplementation can improve the performance of broilers.” was changed to “Vast majority of studies have reported the positive effects of dietary Bacillus subtilis supplementation on the growth performance of broiler chickens” (Line 344-345).

 

Point 7: There are some minor mistakes of English, needs to be corrected. 

 

Response 7: Thank you very much for your excellent comment and suggestion. We have corrected and re-checked the words spelling and grammatical errors of the revised manuscript (Line 42-43, Line 59, Line 187, Line 430).

 

We hope the revised manuscript will meet the requirements. We sincerely appreciate your warm work again.

Please see the attachment.

Author Response File: Author Response.docx