Effects of Peroral Microbiota Transplantation on the Establishment of Intestinal Microorganisms in a Newly-Hatched Chick Model
Round 1
Reviewer 1 Report
The authors used the newborn broiler model to comprehensively analyze the effects of oral microbial transplantation on the intestinal microbiota structure of broilers. These findings have guiding implications for probiotics in the treatment of intestinal health in animal and humans. The topic of this study is basically novel and the research content could well achieve the purpose of study. However, some improvements are needed for the manuscript before being accepted for publication. My suggestions and comments are as follows.
1. Whether the control group also performed oral irrigation and what was used. It needs to be clarified in the material method.
2. The cecum microbiota with no salmonella and the lowest antibiotic resistance was selected as inoculant by detecting antibiotic resistance of kanamycin sulfate, ampicillin sodium, and chloromycetin. How many broilers were used for the collection of cecum microorganisms?
3. Briefly describe the detection methods for Total bacterial, Lactobacillus and Escherichia coli in 2.2.
4. In 2.2, the pellets were collected by centrifuging at 27,000 × g and 4°C for 20 min and then frozen in liquid nitrogen for DNA extraction. Check the centrifugal speed.
5. In 2.2, Information such as temperature and humidity of test room needs to be supplemented. The lighting regime is unreasonable and needs to be verified. Information on raising density such as the number of broilers in one cage and the size of cage needs to be provided.
6. (gauze, 177μm filter cloth, 105 μm filter cloth)。Spaces are required between numbers and units. Check the entire text.
7. By the end, five broilers per replicate were selected and euthanized upon reaching humane endpoints. Provide the specific methods for euthanasia.
8. The title of 2.4 should be corrected as “16S rDNA amplicon pyro-sequencing”
9. In 3.2, Microbiota of inoculant were analyzed by Krona software. Add the version of the software and other information.
10. In 3.4, Chao1, ACE, Simpson, and Shannon, in the control and microbiota transplantation group. An extra comma was used, and check the whole text carefully.
11. In 3.4, it was seen that Aestuariispira, Christensenella, Fervidicella, Gracilibacter, Haloferula, Mycoplasma, Novispirillum and Pantoea were more abundant (p < 0.05) in the microbiota transplantation group than in the control group。”those” is obviously missing behind of ”Than”.
12. In 3.4, This suggested that there was a closely correlation between the ileo-cecal microbiota composition and microbiota transplantation. Correct closely into close.
13. The last paragraph of the discussion should not be limited to poultry industry. it needs to be rewritten. I recommend combining it with the researches related to microbial treatment of human gut health, which has certain guiding significance for the development of probiotic products for humans.
Author Response
Dear reviewer,
Thanks for your constructive comments. We have made detailed modifications carefully to improve our manuscript according to the comments and suggestions. The amendments made to the text are highlighted using the track changes mode in the revised manuscript. Our point-by-point responses are showed as follows. We do hope we could understand your questions correctly and have given right answers in the revised manuscript. Please feel free to inform me if there are still some questions. Thanks for your kind consideration.
Sincerely,
Guohua Liu, Ph.D. Professor
Institute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China
1. Whether the control group also performed oral irrigation and what was used. It needs to be clarified in the material method.
Response: Yes. Same procedure was conducted in the control group using culture medium without microbiota. We supplemented related information in the revised manuscript.
2. The cecum microbiota with no salmonella and the lowest antibiotic resistance was selected as inoculant by detecting antibiotic resistance of kanamycin sulfate, ampicillin sodium, and chloromycetin. How many broilers were used for the collection of cecum microorganisms?
Response: Five broilers were used here. We supplemented the information.
3. Briefly describe the detection methods for Total bacterial, Lactobacillus and Escherichia coli in 2.2.
Response: We supplemented the method as you suggested.
4. In 2.2, the pellets were collected by centrifuging at 27,000 × g and 4°C for 20 min and then frozen in liquid nitrogen for DNA extraction. Check the centrifugal speed.
Response: It is a mistake. We correct it into 2,000 × g.
5. In 2.2, Information such as temperature and humidity of test room needs to be supplemented. The lighting regime is unreasonable and needs to be verified. Information on raising density such as the number of broilers in one cage and the size of cage needs to be provided.
Response: Thanks for your suggestion. There was an hour of darkness every day. We also supplemented the information you mentioned.
6. (gauze, 177μm filter cloth, 105 μm filter cloth)。Spaces are required between numbers and units. Check the entire text.
Response: We corrected it.
7. By the end, five broilers per replicate were selected and euthanized upon reaching humane endpoints. Provide the specific methods for euthanasia.
Response: The birds were euthanized with pentobarbital sodium (100 mg/kg BW) intravenously. We supplemented it the revised manuscript.
8. The title of 2.4 should be corrected as “16S rDNA amplicon pyro-sequencing”
Response: We corrected it.
9. In 3.2, Microbiota of inoculant were analyzed by Krona software. Add the version of the software and other information.
Response: We added it.
10. In 3.4, Chao1, ACE, Simpson, and Shannon, in the control and microbiota transplantation group. An extra comma was used, and check the whole text carefully.
Response: We corrected it.
11. In 3.4, it was seen that Aestuariispira, Christensenella, Fervidicella, Gracilibacter, Haloferula, Mycoplasma, Novispirillum and Pantoea were more abundant (p < 0.05) in the microbiota transplantation group than in the control group. ”those” is obviously missing behind of ”Than”.
Response: We corrected it.
12. In 3.4, This suggested that there was a closely correlation between the ileo-cecal microbiota composition and microbiota transplantation. Correct closely into close.
Response: We corrected it.
13. The last paragraph of the discussion should not be limited to poultry industry. it needs to be rewritten. I recommend combining it with the researches related to microbial treatment of human gut health, which has certain guiding significance for the development of probiotic products for humans.
Response: According to your suggestion, we added a paragraph at the end of the discussion section to discuss the potential probiotics affected by microbiota transplantation.
Reviewer 2 Report
In the manuscript entitled “Effects of peroral microbiota transplantation on the establishment of intestinal microorganisms in a newly-hatched chick model”the author aims to study the gut microbial communities in newly-hatched chicks with and without microbiota transplantation. This study has due importance and the manuscript can be accepted after major revision based on the following comments.
1. In overall manuscript typos and grammatically correction is required such as some of them are mentioned in 2-5
2. In introduction in line 4 “ to against” is incorrect. correct the sentence
3. In introduction 2nd para “big diversity” is used which needs to be replaced by an appropriate word
4. The control group was not given this procedure, but was otherwise treated the same. correct the sentence.
5. In the manuscript in some places “OTU” is written as OUT which needs correction
6. How “inoculants” was stored? was their any difference during its storage for use in two days?
7. From the supplementary tables it seems that 24 samples were processed but in material method 5 birds from each replicates were taken. This point needs proper explanation in material methods, As a whole the material methods needs polishing in terms of explaining sampling and its processing
8. Under the heading “DNA extraction and 16S rDNA amplicon sequencing” DNA extraction is written twice one for genomic and other for microbial? Need clarification.
9. There is no mention of positive or negative controls being carried out during DNA extraction or sequencing.
10. Was the data deposited to any repository? If it is not submitted it submit it and provide the information in “Data Availability Statement”
11. The author has mentioned about the unique and common OTU in the groups at which level (Phylun, genus etc) these unique and common OUT’ s were identified?
12. In the result section under the heading “Alpha diversity and taxonomic composition of microbiota” Figure S2 is mentioned but no representation of diversity indices is found in the figure. The values of indices mentioned in text does not match the values in the tables and the values in text are not understandable. Simspon and Shannon indices values in the table are given twice( both different values) .SEM mentioned is given but which group it represents needs clarification or mention SEM of both groups.
13. Under the heading “Beta diversity, microbiota comparison, and the key species screening” only metabolic pathways specifically carbohydrate metabolism is elaborated while no information on species screening is given.
14. The author has mentioned about beta diversity but failed to explain which beta diversity indices(Jaccard index, Bray Curtis dissimilarity index, Unweighted unifrac metric or weighted unifrac metric) specifically was used for comparison.
15. The author has mentioned about different bacterial counts being done mentioned in table but the unit mentioned (lg/g) refers to what? Abbreviation may be written in complete form in footnote
16. The author has explained the results very well but the results and discussion may be made more appealing if emphasis on explaining some bacterial communities is given which may benefit gut health of the chickens after microbiota transplantation.
Author Response
Dear reviewer,
Thanks for your constructive comments. We have made detailed modifications carefully to improve our manuscript according to the comments and suggestions. The amendments made to the text are highlighted using the track changes mode in the revised manuscript. Our point-by-point responses are showed as follows. We do hope we could understand your questions correctly and have given right answers in the revised manuscript. Please feel free to inform me if there are still some questions. Thanks for your kind consideration.
Sincerely,
Guohua Liu, Ph.D. Professor
Institute of Feed Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China
- In overall manuscript typos and grammatically correction is required such as some of them are mentioned in 2-5
Response: Thank you for your suggestion. We checked the typos and grammar throughout the manuscript.
- In introduction in line 4 “ to against” is incorrect. correct the sentence
Response: We corrected it.
- In introduction 2nd para “big diversity” is used which needs to be replaced by an appropriate word
Response: We corrected it.
- The control group was not given this procedure, but was otherwise treated the same. correct the sentence.
Response: We re-constructed the sentence.
- In the manuscript in some places “OTU” is written as OUT which needs correction
Response: We corrected them.
- How “inoculants” was stored? was their any difference during its storage for use in two days?
Response: Stored in 4°C. We supplemented the information. It was no difference for use in two days.
- From the supplementary tables it seems that 24 samples were processed but in material method 5 birds from each replicates were taken. This point needs proper explanation in material methods, As a whole the material methods needs polishing in terms of explaining sampling and its processing
Response: We are sorry for the misunderstanding. In fact, 5 birds were selected from the each replicate and their microbial DNA samples were mixed in equal amounts to generate one sample. One treatment contained 6 replicates, so a total of 6 samples were taken for one treatment. We improved the description in the manuscript.
- Under the heading “DNA extraction and 16S rDNA amplicon sequencing” DNA extraction is written twice one for genomic and other for microbial? Need clarification.
Response: It is a mistake. We corrected it.
- There is no mention of positive or negative controls being carried out during DNA extraction or sequencing.
Response: Thanks for your reminding. We supplemented the description about the blank control.
- Was the data deposited to any repository? If it is not submitted it submit it and provide the information in “Data Availability Statement”
Response: Thanks for your suggestion. We will submit it a repository and provide the information in “Data Availability Statement” before publication.
- The author has mentioned about the unique and common OTU in the groups at which level (Phylun, genus etc) these unique and common OTU’ s were identified?
Response: The unique and common OTU in the groups is identified by compared with the two experimental groups. The microbiota was recognized base the information of OTU.
- In the result section under the heading “Alpha diversity and taxonomic composition of microbiota” Figure S2 is mentioned but no representation of diversity indices is found in the figure. The values of indices mentioned in text does not match the values in the tables and the values in text are not understandable. Simspon and Shannon indices values in the table are given twice( both different values) .SEM mentioned is given but which group it represents needs clarification or mention SEM of both groups.
Response: We are sorry for the mistake. We corrected it in the main text and detailed the meaning of SEM in all table notes. The data in Table 3 includes two parts, cecum and ileum.
- Under the heading “Beta diversity, microbiota comparison, and the key species screening” only metabolic pathways specifically carbohydrate metabolism is elaborated while no information on species screening is given.
Response: According to your suggestion, we supplemented related information and re-constructed the result section.
- The author has mentioned about beta diversity but failed to explain which beta diversity indices(Jaccard index, Bray Curtis dissimilarity index, Unweighted unifrac metric or weighted unifrac metric) specifically was used for comparison.
Response: In the current study, we conducted PCA and PLS analysis to evaluate the β-diversity of microbiota. The difference of species abundance distribution between samples can be quantitatively analyzed by distance in statistics. Statistical algorithms Euclidean, Bray-Curtis, Unweighted_unifrac and unweighted_unifrac are used to calculate the distance between pairs of samples and obtain the distance matrix. These indices you mentioned are statistical algorithms for beta diversity analysis and visual statistical analysis.
- The author has mentioned about different bacterial counts being done mentioned in table but the unit mentioned (lg/g) refers to what? Abbreviation may be written in complete form in footnote
Response: lg represents the logarithmic value in base 10. We defined it in the footnote.
- The author has explained the results very well but the results and discussion may be made more appealing if emphasis on explaining some bacterial communities is given which may benefit gut health of the chickens after microbiota transplantation.
Response: Thanks for your suggestion. We added a paragraph at the end of the discussion section to discuss the potential probiotics affected by microbiota transplantation.
Round 2
Reviewer 2 Report
Although, manuscript is improved, it still think authors should submit the data in open repositories and add the relevant information in manuscript before being accepted for publication.
Author Response
Dear reviewer,
We have uploaded the raw data in NCBI according to your suggestion. Thanks for your help.
