Identification of Enterotype and Its Effects on Intestinal Butyrate Production in Pigs
Round 1
Reviewer 1 Report
Much improved work although in general there are some bugs in a way that would greatly improve the work, especially special interest in the results (tables and figures).
- You do not put an abbreviation index; I think it could help you understand the text.
- The best way to define the abbreviations is: Enterotype (ET).
- P_values are not well defined.2888E-10 = <0.0001. Please, review all P_Values.
- Figure and Table 2 are in the same line, please change it.
- Tables and Figures, you should put all units. OUT for example. Please change it.
- Form errors. Figure 2, please check the format.
- Figure 2: For example, you should explain, if it is a scatter plot, a confidence interval etc.(Fig 2, 3 and 5).
- Regarding results, usually a is minor than b and b minor than c, please check it.
- In Figure 5 is R or R2?
Author Response
You do not put an abbreviation index; I think it could help you understand the text.
The best way to define the abbreviations is: Enterotype (ET).
Response: Thanks for your suggestion. An abbreviation index was included in Line 347 of the revised manuscript.
P_values are not well defined.2888E-10 = <0.0001. Please, review all P Values.
Response: We have corrected that in the manuscript.
Figure and Table 2 are in the same line, please change it.
Response: We have corrected that.
Tables and Figures, you should put all units. OUT for example. Please change it.
Response: Thanks for your suggestion. We have double checked the tables and figures and added the units accordingly.
Form errors. Figure 2, please check the format.
Response: “OTU” has been changed to “Number of OTUs” in Figure 2 of the revised manuscript.
Figure 2: For example, you should explain, if it is a scatter plot, a confidence interval etc.(Fig 2, 3 and 5).
Response: Thanks for your suggestion. The words “with the 95% confidence interval” have been added to the Figure 2, 3, 5, and 6. Please find them in the revised manuscript.
Regarding results, usually a is minor than b and b minor than c, please check it.
Response: Thanks for your suggestion. We have made changes in the Table 2, Figure 2, 3, 5, and 6 in the revised manuscript.
In Figure 5 is R or R2?
Response: It is R in Figure 5. R is commonly used in the figure of correlation analysis, for examples, in the Figure 1 of Jiang et al. (2015) and in the Figure 4 of Yuan et al. (2020).
Jiang C, Xie C, Lv Y, et al. Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction. Nature Communications, 2015, 6, 10166.
Yuan Z, Yan W, Wen C, et al. Enterotype identification and its influence on regulating the duodenum metabolism in chickens. Poultry Science, 2020, 99, 1515-1527.
Reviewer 2 Report
As I reviewed this manuscript before, I am concerned the study population and sample collection in this study.
And the concerns are still not cleared yet, completely. Even the authors respond to the concerns, livestock animals could be better and thoroughly controlled in genetic and environmental conditions compare with the human. That being so, the animal study using a same sample size can provide more relevance results to compare with the human study. Please consider of my this concern and mind. I am very appreciated to your kind remind.
Author Response
And the concerns are still not cleared yet, completely. Even the authors respond to the concerns, livestock animals could be better and thoroughly controlled in genetic and environmental conditions compare with the human. That being so, the animal study using a same sample size can provide more relevance results to compare with the human study. Please consider of my this concern and mind. I am very appreciated to your kind remind.
Response: Thanks for your comments. All the experimental pigs were purebred Jinhua pigs and housed in the same environmentally controlled pens. Because the aim of this study was to investigate the enterotype and its effects on intestinal butyrate production in pigs, we didn’t compare with human study but we referred to the ideas of previous human and animal study about enterotype. Thanks again. We will do as your suggestion in the future research.
Reviewer 3 Report
Authors adequately answer to the questions raised
Author Response
Thanks for your previous suggestion.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The article by Xu et al titled “Identification of enterotype and its effects on intestinal Butyrate Production in Pigs” attempting to identify enterotypes and establishing a relation between the identified enterotypes and butyrate production in Jinhua is a promising field of research. The article is helpful in understanding the relationship between enterotypes clustering and butyrate production. However, there are certain remarks that authors should address.
-Line 31: Please remove the words “but the order was not the same”.
-Line 38: Please replace the words “than in those of the” with words “as compared to”.
-Line 43: Please replace the words “were of” and “than they were in” with words “showed” and “as compared to” respectively.
-Line 54: Please remove “which provides numerous biological activities that the host lacks”.
-Line 57: Please remove the “correspondingly,”.
-Line 58: Please replace the word “nutrients” with the word “metabolites”.
-Line 70: Please remove the word “agricultural”.
-Line 88: Please remove “whether the ET clustering depends on the pig breeds has been seldom studied.”
-Lines 93 & 94: Please remove “The findings will provide a new perspective to understand the effect of the gut microbiota on the nutrient metabolism in pigs.”
-Line 136: Please clarify “ten times (mL) Deionized Water”.
-Line 156: Please reconsider the words “less than 0.05” in the sentence “A P-value of less than 0.05 was considered statistically significant” and clarify whether they can be replaced with “equal to or greater than 0.05”.
-Lines 192 & 193: Please rewrite the sentence “The relative abundance 192 of the most dominant bacterial genus in each ET was significantly higher than that in the other 2 ETs.” for better understanding.
-Lines 221-224: Please clarify the sentence “Consistent with the aforementioned results, significant more copies of the terminal genes for butyrate synthesis, butyrate kinase (Buk) and butyryl coenzyme A (CoA): acetate CoA transferase (But) in the fecal samples of the ET3 group than those of the other 2 groups (Figure 5B and C).”
-Line 265: Please replace the words “the different” with the words “variations in”.
-Line 277: Please replace the word “period” with the word “phase”.
-Line 278 & 279: Please remove the sentence “In the future study, more gut microbiome samples should be included across 278 different age strata to get a full view of the ET clustering and switching in Jinhua pigs.”
-Line 291: Please replace the word “fermenting” with the word “ferment”.
Author Response
We highly appreciate all your comments and have improved our manuscript accordingly.
--Please remove the words “but the order was not the same”.
Response: Done. Please see L33.
--Line 38: Please replace the words “than in those of the” with words “as compared to”.
Response: Done. Please see L39-40.
--Line 43: Please replace the words “were of” and “than they were in” with words “showed” and “as compared to” respectively.
Response: Done. Please see L44-45.
--Line 54: Please remove “which provides numerous biological activities that the host lacks”.
Response: Done. Please see L54.
--Line 57: Please remove the “correspondingly,”.
Response: Done.
--Line 58: Please replace the word “nutrients” with the word “metabolites”.
Response: Done. Please see L57.
--Line 70: Please remove the word “agricultural”.
Response: Done.
--Line 88: Please remove “whether the ET clustering depends on the pig breeds has been seldom studied.”
Response: Done.
--Lines 93 & 94: Please remove “The findings will provide a new perspective to understand the effect of the gut microbiota on the nutrient metabolism in pigs.”
Response: Done. Please see L92.
--Line 136: Please clarify “ten times (mL) Deionized Water”.
Response: Thanks for your comment. We have changed the words “ten times (mL) Deionized Water” into “1 mL deionized water”.
--Lines 192 & 193: Please rewrite the sentence “The relative abundance of the most dominant bacterial genus in each ET was significantly higher than that in the other 2 ETs. ” for better understanding.
Response: Thanks for your comment. We have removed these words because it indicated little information and may be misleading.
--Lines 221-224: Please clarify the sentence “Consistent with the aforementioned results, significant more copies of the terminal genes for butyrate synthesis, butyrate kinase (Buk) and butyryl coenzyme A (CoA): acetate CoA transferase (But) in the fecal samples of the ET3 group than those of the other 2 groups (Figure 5B and C).”
Response: This sentence has been rewritten. Please find them in Lines 238-244 of the revised manuscript.
--Line 265: Please replace the words “the different” with the words “variations in”. Response: Done. Please see L286.
--Line 277: Please replace the word “period” with the word “phase”.
Response: Done. Please see L298.
--Line 278 & 279: Please remove the sentence “In the future study, more gut microbiome samples should be included across different age strata to get a full view of the ET clustering and switching in Jinhua pigs.”
Response: Done.
--Line 291: Please replace the word “fermenting” with the word “ferment”.
Response: Done. Please see L311.
Reviewer 2 Report
General comments:
Please use an index of abbreviations used, it would be much easier to read.
Some abbreviations used are not well identified, for example, ET of the simple summary, is not defined. Please review all. (line 15)
Introduction:
No specific comments.
Material and methods:
Line 100: How many animals have they used? It should be specified for each analysis and for each treatment. At what point were these samples taken?
Statistics.
Line 153: The p_values as they were determined? What result is there of normality? Can those analyzes be used? Outlayers study?
Results:
General comments to tables and figures:
Tables and figures must be self explanatory. ET1, ET 2 and ET3 should specify what they are.
Explanation is lacking, above all, of certain graphs where it is not clear what each thing means. For example, explain, if it is a scatter plot, a confidence interval etc.(Fig 2, 3 and 5).
Figure 2:
Line 204: Why do not the different letters appear here as will be done later?.
Table 1 (2):
Line 205: There are two tables one. They must modify the name
Why is the p_value expressed with those decimals? Normally you use another nomenclature
Line 205: Why doesn't the sem appear?
Figure 3:
Line 207: Sometimes they put the largest letter with alphabetical consequence eg. C> B>A but other times not. They should unify criteria.
Table 2:
Line 228: Check the decimals, the logical thing would be to put more when the units are smaller.
Figure 5:
Line 232: In the definition of the figure, different letters (A,B and C) appear that do not correspond to the figure.
Figure 6:
Line 250: Sometimes there is too much flexibility in some treatment. To what is due? Have you checked outlayers? Have they verified that the proposed statistical approach can be carried out?
Conclusions:
Line 314 and 322: Please, do not use abbreviations in conclusion.
Author Response
General comments:
Please use an index of abbreviations used, it would be much easier to read.
Some abbreviations used are not well identified, for example, ET of the simple summary, is not defined. Please review all. (line 15)
Response: Thanks for your suggestion. We have checked the manuscript and defined the abbreviations.
Introduction:
No specific comments.
Material and methods:
Line 100: How many animals have they used? It should be specified for each analysis and for each treatment. At what point were these samples taken?
Response: We have rewritten the “Animals and Sample Collection” using the following words:
The animals and fecal sample collection were described in our previous report [33]. Briefly, a total of 105 female Jinhua pigs were used in this study. The experimental pig cohort were from 29 Jinhua sows which housed in a farrowing house. Pigs were kept in the environmentally controlled facility after weaning, where 5-8 pigs were housed per pen, and raised under the same commercial feed based on corn and soybean meal and management manners. Fresh fecal samples were obtained at 105 days of age and stored at -80 °C until analysis.
Please find them in Lines 98-101 in the revised manuscript.
Statistics.
Line 153: The p values as they were determined? What result is there of normality? Can those analyzes be used? Outlayers study?
Response: Thanks for your comments. We have double checked the Statistic and found there were some mistakes regarding the description of the statistic method that we used. Kruskal–Wallis test was used for the statistical analyses to test the differences in alpha diversity, the relative abundance of microbial taxa, and the butyric acid concentrations and butyrate synthesis gene copies among ETs. We have rewritten these sentences as following:
Statistical analyses and graphing were conducted using SPSS statistics software (version 20.0) and Graphpad Prism Program (version 6.0), respectively. Data are expressed as means±SEM. The Kruskal-Wallis test was used to compare the α-diversities and the relative abundance of microbial taxa among ETs. The comparison analysis of the butyrate concentrations and butyrate synthesis gene copies (Buk and But) among ETs was performed using the Kruskal–Wallis test [23,27]. Spearman correlation coefficient was used to describe the co-occurrence patterns among the predominant genera and the relationships between relative abundances of butyrate-producing bacteria, fecal contents of butyrate, and gene copies of Buk and But. Significance was set at P < 0.05.
Results:
General comments to tables and figures:
Tables and figures must be self explanatory. ET1, ET 2 and ET3 should specify what they are.
Explanation is lacking, above all, of certain graphs where it is not clear what each thing means. For example, explain, if it is a scatter plot, a confidence interval etc.(Fig 2, 3 and 5).
Response: Thanks for your comments. We have rewritten the titles in Figures and Tables and added the information to specify what they are.
Figure 2:
Line 204: Why do not the different letters appear here as will be done later?.
Response: We have made changes for Figure 2 in the revised manuscript as you suggested.
Table 1 (2):
Line 205: There are two tables one. They must modify the name
Response: Done. Table1-4 are listed in the revised manuscript.
Why is the p_value expressed with those decimals? Normally you use another nomenclature
Line 205: Why doesn't the sem appear?
Response: We have made changes for Table 2 in the revised manuscript as you suggested.
Figure 3:
Line 207: Sometimes they put the largest letter with alphabetical consequence eg. C> B>A but other times not. They should unify criteria.
Response: Thanks for your comments. We check the Instructions for Authors of Animals (the Journal belongs to MDPI). It is ok to indicate a significant difference with different letters as it is now. The following is some papers published by Animals, in which significant difference was shown just by the different letters:
[1] Hamzaoui, S.; Caja, G.; Such, X.; Albanell, E.; Salama, A.A.K. Effect of Soybean Oil Supplementation on Milk Production, Digestibility, and Metabolism in Dairy Goats under Thermoneutral and Heat Stress Conditions. Animals, 2021, 11, 350.
[2] Du, X.; Xiang, Y.; Lou, F.; Tu, P.; Zhang, X.; Hu, X.; Lyu, W.; Xiao, Y. Microbial Community and Short-Chain Fatty Acid Mapping in the Intestinal Tract of Quail. Animals, 2020, 10, 1006.
Table 2:
Line 228: Check the decimals, the logical thing would be to put more when the units are smaller.
Response: Thanks for your comments. We check the Instructions for Authors of Animals (the Journal belongs to MDPI). It is ok to have four decimals for the values in Table.
Figure 5:
Line 232: In the definition of the figure, different letters (A, B and C) appear that do not correspond to the figure.
Response: We have made changes for Figure 5 in the revised manuscript.
Figure 6:
Line 250: Sometimes there is too much flexibility in some treatment. To what is due? Have you checked outlayers? Have they verified that the proposed statistical approach can be carried out?
Response: It is normal that the intestinal community structure varied greatly in human and animals. We used the Kruskal-Wallis test to compare the difference the relative abundance of microbial taxa among ETs. Yuan et al. (2020) and Lu et al. (2020) also employed the Kruskal-Wallis test to analyze the difference of microbial taxa. You can find these two references in the manuscript.
[1] Yuan, Z.; Yan, W.; Wen, C.; Zheng, J.; Yang, N.; Sun, C. Enterotype identification and its influence on regulating the duodenum metabolism in chickens. Poult. Sci. 2020, 99, 1515‐1527.
[2] Lu, D.; Tiezzi, F.; Schillebeeckx, C.; McNulty, N. P.; Schwab, C.; Shull, C.; Maltecca, C. Host contributes to longitudinal diversity of fecal microbiota in swine selected for lean growth. Microbiome 2018, 6, 4.
Conclusions:
Line 314 and 322: Please, do not use abbreviations in conclusion.
Response: We have made changes for the abbreviations in conclusion as you suggested.
Reviewer 3 Report
Dear Authors,
This study identified the clearly comparted three enterotypes (ETs) from the fecal microbiome data and their different roles in possible digestion regarding with the fatty acids from GIT. However, the current study design has obvious lacks or limitations to support their hypothesis. The Jinhua breeding pigs were used as a study population. The pigs population has presumably complex family structure and particularly in dam lines. The most enterotypes in swine farms could be strongly connected with their maternal environment during their lactation period. Therefore, the current description for their raising condition using only same commercial feed could not make sure that the feed and genetic environments including genders were appropriately controlled. Moreover, the method for individual fecal samples was not satisfied avoid every possible contaminations among the pigs in the farm. Mirobial populations can be very changeable according to the fluid environments. Therefore, the microbiome study from the GIT must be controlled and exactly targeted (i.e. intestinal microbiome or gut microbiome as in the specific environmental condition). If not, the cohort population study like the current design should provide more details as much as possible and very carefully cut off or choose the selective individuals among the entire population. In the end, it is hard to agree that the identified three ETs in this study are representative microbial groups in the GIT of Jinha pigs and the ETs have important roles in the digestion from the only fecal samples.
All the best,
Author Response
Thanks for your comments and constructive suggestions for us to improve our manuscript. We have rewritten the Animals and Sample Collection and provided the details of experimental pigs and sampling in the revised manuscript.
As you mentioned that The pig population has presumably complex family structure and particularly in dam lines. The most enterotypes in swine farms could be strongly connected with their maternal environment during their lactation period.
Response: All the experimental pigs were from the purebred Jinhua sows which housed in the same environmentally controlled farrowing house. In that case, the environment was same for the piglets during their lactation period.
As for your question regarding the effect of genetic environments of sow on the enterotype. Our interpretation is as following:
The aim of this study was to investigate the enterotype and its association with intestinal butyrate production in pigs. Although the Jinhua pig population from different sows, it is commonly that the gut microbiota processes the indigestible polysaccharides in the diet to produce butyrate. We read some references about the pig enterotype, and found that the authors didn’t think the pig family structure having an effect on the enterotype (Ramayo-Caldas et al., 2016; Yang et al., 2017; Lu et al., 2018; Le Sciellour et al., 2019).
Additionally, when investigating the enterotypes of human gut microbiota, Arumugam et al (2011) used the human data from different countries, ethnicities, and ages. Therefore, the scientists pay much attention to the enterotype, mainly analyzing the function variation between enterotypes rather than the relationship between genetic background and enterotype.
[1] Yuliaxis Ramayo-Caldas, Nuria Mach, Patricia Lepage, et al. Phylogenetic network analysis applied to pig gut microbiota identifies an ecosystem structure linked with growth traits. The ISME Journal (2016) 10, 2973–2977
[2] Yang, H.; Huang, X.; Fang, S.; He, M.; Zhao, Y.; Wu, Z.; Yang, M.; Zhang, Z.; Chen, C.; Huang, L. Unraveling the fecal microbiota and metagenomic functional capacity associated with feed efficiency in pigs. Front Microbiol. 2017, 8, 1555.
[3] Lu, D.; Tiezzi, F.; Schillebeeckx, C.; McNulty, N. P.; Schwab, C.; Shull, C.; Maltecca, C. Host contributes to longitudinal diversity of fecal microbiota in swine selected for lean growth. Microbiome 2018, 6, 4.
[4] Le Sciellour, M.; Renaudeau, D.; Zemb, O. Longitudinal analysis of the microbiota composition and enterotypes of pigs from post-eeaning to finishing. Microorganisms 2019, 7, 622.
[5] Arumugam, M.; Raes, J.; Pelletier, E.; Le Paslier, D.; Yamada, T.; Mende, D.R. Enterotypes of the human gut microbiome. Nature 2011, 473, 174–180.
As you mentioned that the current description for their raising condition using only same commercial feed could not make sure that the feed and genetic environments including genders were appropriately controlled.
Response: In order to ensure that the raising condition could be controlled, all pigs were kept in the environmentally controlled facility, where 5-8 pigs were housed per pen.
The experimental pigs used in this study were all female. The Jinhua pig is a well-known indigenous breed of Zhejiang Province in eastern China with a slow growth rate but a high propensity for adipogenesis and intramuscular fat deposition. The female Jinhua pigs were commonly used as sows to produce more purebred Jinhua pigs or to corssbreed with boar Duroc, Landrace or Yorkshire to improve the production performance and the intramuscular fat. So it would be more significant to study the microbial community structure and enterotype associated with the butyrate production in female pigs. Therefore, we collected the fresh feces from the female Jinhua pigs in this study.
As you mentioned that the method for individual fecal samples was not satisfied avoid every possible contaminations among the pigs in the farm.
Response: We cleared the floor of pens before sample collection. There were some students stayed in the pen to wait for the fresh feces excreted by pigs. Once the pigs defecated, the part of fresh feces without contacting the floor were collected. So we thought that there was little contamination among the pigs in the farm.
Reviewer 4 Report
The present study is interesting, in line with many attempts made to understand the microbiota contribute to general health.
The point that need to be introduced in the present study is the lack of reference to sex differences. Authors never mention the sex of the animals tested and, due to the note about the age, it became importat to know if the SD values are due to the sex of the animal because in the age taken into account there is the development of the two sex, differently!
Author Response
The present study is interesting, in line with many attempts made to understand the microbiota contribute to general health.
The point that need to be introduced in the present study is the lack of reference to sex differences. Authors never mention the sex of the animals tested and, due to the note about the age, it became importat to know if the SD values are due to the sex of the animal because in the age taken into account there is the development of the two sex, differently!
Response: A total of 105 female Jinhua pigs were used in this study.
The Jinhua pig is a well-known indigenous breed of Zhejiang Province in eastern China with a slow growth rate but a high propensity for adipogenesis and intramuscular fat deposition. The female Jinhua pigs were commonly used as sows to produce more purebred Jinhua pigs or to corssbreed with boar Duroc, Landrace or Yorkshire to improve the production performance and the intramuscular fat. So it would be more significant to study the microbial community structure and enterotype associated with the butyrate production in female pigs. Therefore, we collected the fresh feces from the female Jinhua pigs in this study.
