Identification of Gut Microbiota Affecting Fiber Digestibility in Pigs

Round 1

Reviewer 1 Report

The manuscript cimb 1905843 entitled “ Identification of Gut Microbiota Affecting Fiber Digestibility in pig” reports the faecal bacterial differences related with fiber digestibility in pigs. The authors explored the functional composition of that bacterial community by using the available software PICRUSt what in a certain way is limited. Several comments/suggestions in order to improve the manuscript are indicated below.

 

Abstract: The differences in the microbial composition between groups is required first and then indicate the associated metabolic pathway differences.

Line 61 change to It was showed that the genera

Line 61 Each genus name is in italic

Line 63-65. this sentence is not clear. Revise it and rewrite.

Line 68-69 But what are the main characteristics regarding fiber digestibility of the Suhuai pig? In what is different from the other breeds?

Line 93 16S rRNA in italic. Revise and change all over the manuscript.

Line 103 AIA in full first.

Line 105 correction required

Line 112-119. Rewrite this section. It is not indicated what kind of sequencing process was performed. An appropriate description is required.

Line 133 Change the bacteria enriched to the enriched bacteria

Line 141 change were shown to are shown. Revise this all over the manuscript; the authors are showing the data not showed

Line 142 CV in full first

Line 142 Indicate first the mean +- the SE. And about statistics?

Line 160 Simpson with first capital letter.

Line 162 mean A-D or just A and C? Why not use the same colour for each group in order to facilitate the analysis by the reader?

Line 162-165 here and all over the manuscript: Revise the scientific names, which is mandatory to be in italic.

Line 162-165 change to: Firmicutes and Bacteroidetes were the most predominant phyla in all samples comprised more than 91% of the total sequences.

Line 168 change In the meantime to In contrast

Line 169-170 check: Fibrobacter or Fibrobacteres? Phylum or genus?

Line 183 I cannot see the genus Lachonospira in Figure S2 D, instead it is indicated the group of the Lachnospiraceaefamily, what is not the same.

Line 199 I do not see genera, but members of the different phyla and families. Correction required. This inaccuracy is all over the manuscript. Use the appropriate terms.

Line 253-257 This is repetition of data. Rewrite

Line 270 not microbial! but bacterial. the authors used 16SrRNA gene!

Line 277 about Lachnospira see the above comment.

Line 283 Ruminococcaceae is not a genus but a family, what is indicated can be a member of this family. Rewrite according.

Line 289 change complicated to complex.

Line 291-292 Change: The data between the two types of samples analyzed by Adonis/PERMANOVA showed statistical significance. 

What were the two types of samples? More detail about this citation is required.

Line 297 shannon and simpson -initial letters are capital

Line 300  a link to the internet is found here :microbialcommunitystructure. Eliminate and correct

Line 310-311 It is plausible to use 29 or 23 genera as biomarkers? This has to be discussed realistically.

Line 319 Marques: also here there is a link. Eliminate it and add et al

Line 321-329. Just describing the data of other studies without proper discussion is not acceptable. Rewrite adding substantial analysis.

 

Author Response

1. - Abstract: The differences in the microbial composition between groups is required first and then indicate the associated metabolic pathway differences.

RE: Thank you for the comments which are very helpful to make this revision. We completely agree with you. “The most important functions of the above different potential biomarkers were Carbohydrate transport and metabolism, General function prediction only, Amino acid transport and metabolism, Cell wall/membrane/envelope biogenesis, Translation, Transcription, Replication, Energy production and conversion, Signal transduction mechanisms and Inorganic ion transport and metabolism. The most important metabolic pathways of the above different potential biomarkers Membrane transport, Carbohydrate metabolism, Amino acid metabolism, Replication and repair, Translation, Cell motility, Energy metabolism, Poorly characterized, Nucleotide metabolism, Metabolism of cofactors and vitamins and Cellular processes and signaling.” has been added. Line 32-40

2. Line 61 change to It was showed that the genera

RE: This is a basic grammar mistake we've made. Thank you so much for mentioned it. “the genera” has been added. Line 67-68

3. Line 61 Each genus name is in italic

RE:  It’s really our carelessness and it has been revised. Line 68-69

4. Line 63-65. this sentence is not clear. Revise it and rewrite.

RE: Thank you very much for your reminding. It has been rewritten as “As the gut bacteria related to fiber digestibility are difficult to be isolated and cultured in a traditional way, more microbiota associated with NDF and ADF digestibility need to be further verify and discover with 16S rRNA gene sequencing technology”. Line 70-73

5. Line 68-69 But what are the main characteristics regarding fiber digestibility of the Suhuai pig? In what is different from the other breeds?

RE: Thank you so much for your questions and these are very important points. Chinese indigenous pig breeds had good performance in crude fiber digestion. Chinese Suhuai pig is a new national lean-type pig breed, as containing 25% Chinese indigenous Huai pig ancestry and 75% Large White ancestry, possesses strong ability to digest high fibrous feedstuffs. And we also could obtained samples with low fiber digestive capacity. Therefor Suhuai pigs were chosen to be study in the present study.

6. Line 93 16S rRNA in italic. Revise and change all over the manuscript.

RE: Thank you for the suggestion. It has been revised. Line 101

7. Line 103 AIA in full first.

RE: Thank you for your reminding. “Acid insoluble ash (AIA) was used as an indigestible marker to assess the digestibility of the dietary components (AOAC 942.05)” has been added. Line 107-108

8. Line 105 correction required

RE: Thank you for your reminding, it has been revised in the manuscript. Line 114

9. Line 112-119. Rewrite this section. It is not indicated what kind of sequencing process was performed. An appropriate description is required.

RE: Thank you for the suggestion. It has been substituted by “The barcoded fusion forward primer was 520F (5’-GCACCTAAYTGGGYDTAAAGNG-3’), the reverse primer was 802R (5’-TACNVGGGTATCTAATCC-3’). The PCR condition was as follows: initial denaturation at 98℃ for 5 min; 98℃ denaturation for 10 sec, 50℃ annealing for 30 sec, and 72℃ extension for 30 sec, repeated for 25 cycles; fifinal extension at 72℃ for 5 min. All samples were amplified in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and quantified using Quantus^TM Fluorometer (Promega, USA).” Line 121-131

10. Line 133 Change the bacteria enriched to the enriched bacteria

RE: Thank you for the advice. We change Linear discriminate analysis effect size (LEfSe)

analysis to groups comparison analyses with Wilcoxon rank-sum test as other reviewer suggested. Line 144-145

11. Line 141 change were shown to are shown. Revise this all over the manuscript; the authors are showing the data not showed

RE: Thank you for your reminding, this was our mistake in writing. They have been revised. Line 153, 247, 249

12. Line 142 CV in full first

RE: Thank you for your reminding. “variable coefficient (CV)” has been added.  Line 155

13. Line 142 Indicate first the mean +- the SE. And about statistics?

RE: Thank you for the advice. “The mean ± the SE of ATTD NDF was 70.4±0.6 and that of ATTD ADF was 62. 9±1.2.” has been added. Line 154

Results analyzed by SAS software are presented as means, standard errors and coefficients of variation.

14. Line 160 Simpson with first capital letter.

RE: Thank you for your reminding. It has been revised. Line 174

15. Line 162 mean A-D or just A and C? Why not use the same colour for each group in order to facilitate the analysis by the reader?

RE: Thank you for the advice. Figure S2 is showed Phyla and genera distribution. Same colour has been used for each group. Phyla and genera distributions as a percentage of the total number in NDF (A and C) and ADF (B and D) groups, respectively. Supplemental material

16. Line 162-165 here and all over the manuscript: Revise the scientific names, which is mandatory to be in italic.

RE: Thank you so much for your reminding. The scientific names have been in italic all over the manuscript. Line 176-181

17. Line 162-165 change to: Firmicutes and Bacteroideteswere the most predominant phyla in all samples comprised more than 91% of the total sequences.

RE: Thank you for the advice, and it has been revised. Line 179-180

18. Line 168 change In the meantime to In contrast

RE: Thank you for the suggestion. It has been changed. Line 182

19. Line 169-170 check: Fibrobacter or Fibrobacteres? Phylum or genus?

RE: Thank you for the thoughtful suggestion. Sequences were classified to Fibrobacteres and it is a phyla.

20. Line 183 I cannot see the genus Lachonospira in Figure S2 D, instead it is indicated the group of the Lachnospiraceae family, what is not the same.

RE: Thank you so much for your kind comments on our errors in the date and we have checked carefully. These mistakes have been revised to “The 2 most predominant genera in H- and L-AD of ADF, separately containing about 26.7% and 8.4% of the total sequences, were Lactobacillus and Christensenellaceae_R-7 group also belong to the phylum Firmicutes (Figure S2 D).” Line 195-198

21. Line 199 I do not see genera, but members of the different phyla and families. Correction required. This inaccuracy is all over the manuscript. Use the appropriate terms.

RE: Thank you for the important advice. We considered another way of representation about figure 3 as other reviewer suggested.

In this study taxonomy was assigned using the Silva (Release128 http://www.arb-silva.de), a total of 190 genera were identified. norank_f_p-2534-18B5_gut_group and norank_f_Porphyromonadaceae were classified to genera. In this manuscript, 30 and 29 groups of sequences were classified to genera (table 1 and table 2), which were associated with ADDT of NDF and ADF. Some papers also classified unclassifified_o_Lactobacillales and norank_f_Bacteroidales_S24-7_group to genera (Pu, G.2022; Liu, B. 2021)

Table 1 Thirteen genera were found to be associated with ADDT of NDF.

 

 

 

H-AD of NDF		L-AD of NDF
Anaeroplasma	Prevotella_1	Lactobacillus
Anaerovibrio	Ruminiclostridium_1
Candidatus_Soleaferrea	Ruminococcaceae_NK4A214_group
dgA_11_gut_group	Ruminococcaceae_UCG_004
Erysipelotrichaceae_UCG_003	Ruminococcaceae_UCG_005
Eubacterium__coprostanoligenes_group	Ruminococcus_1
Family_XIII_AD3011_group	Staphylococcus
Lachnospiraceae_ND3007_group	Succinivibrio
Lachnospiraceae_NK4A136_group	Treponema_2
Lachnospiraceae_NK4B4_group	unclassified_f__Lachnospiraceae
norank_f__Bacteroidales_S24_7_group	unclassified_f__Ruminococcaceae
norank_f__Mitochondria	unclassified_k__norank
norank_f__p_2534_18B5_gut_group	unclassified_o__Bacteroidales
norank_f__Porphyromonadaceae	unclassified_p__Bacteroidetes
Oscillibacter

H-AD of ADF		L-AD of ADF
Candidatus_Soleaferrea	Prevotella_2	Clostridium_sensu_stricto_6
dgA_11_gut_group	Prevotellaceae_UCG_001	Lachnospira
Eubacterium__coprostanoligenes_group	Quinella	Lachnospiraceae_FCS020_group
Family_XIII_AD3011_group	Ruminococcaceae_NK4A214_group	Lachnospiraceae_UCG_007
Fibrobacter	Ruminococcaceae_UCG_002	p_1088_a5_gut_group
norank_c__WCHB1_41	Schwartzia	Ruminococcus_2
norank_f__Clostridiales_vadinBB60_group	Succinivibrio
norank_f__p_2534_18B5_gut_group	unclassified_f__Ruminococcaceae
norank_f__Porphyromonadaceae	unclassified_k__norank
norank_o__Bradymonadales	unclassified_o__Clostridiales
Oscillibacter

Table 2 Twenty nine genera were found to be associated with ADDT of ADF.

 

References are as follows.

Pu, G.; Hou, L.; Du, T.; Wang, B.; Liu, H.; Li, K.; Niu, P.; Zhou, W.; Huang, R.; Li, P. Effects of short-term feeding with high fiber diets on growth, utilization of dietary fiber, and microbiota in pigs. Frontiers in microbiology 2022, 13, 963917.

Liu, B.; Zhu, X.; Cui, Y.; Wang, W.; Liu, H.; Li, Z.; Guo, Z.; Ma, S.; Li, D.; Wang, C.; Shi, Y. Consumption of dietary fiber from different sources during pregnancy alters sow gut microbiota and improves performance and reduces inflammation in sows and piglets. mSystems 2021, 26, 6

22. Line 253-257 This is repetition of data. Rewrite

RE: Thank you so much for the important reminds. “We hypothesized that there was variation in fiber digestibility in Suhuai pig breed and we found that coefficients of variation of ADDT NDF and ADF in Suhuai Pigs were 12.08 and 18.80%, respectively.” has been deleted.

23. Line 270 not microbial! but bacterial. the authors used 16SrRNA gene!

RE: Thank you for the advice. It has been revised. Line 274

24. Line 277 about Lachnospira see the above comment.

RE:  Thank you again for your kind comments on our errors in the date and this mistake has been revised. Line 286

25. Line 283 Ruminococcaceae is not a genus but a family, what is indicated can be a member of this family. Rewrite according.

RE: Thank you for the important advice. Ruminococcaceae absolutely is a family, however, Ruminococcaceae_UCG-005 was classified to a genus via Silva database. We found several researches had similar classified results. In previous study, sequences were classified to Ruminococcaceae_UCG-005 and Prevotellaceae_NK3B31_group on genera level by Pu et al. In Boshuai Liu et al study, the dominant genera included Christensenellaceae_R-7_group and Christensenellaceae is a family.

References are as follows.

Pu, G.; Hou, L.; Du, T.; Wang, B.; Liu, H.; Li, K.; Niu, P.; Zhou, W.; Huang, R.; Li, P. Effects of short-term feeding with high fiber diets on growth, utilization of dietary fiber, and microbiota in pigs. Frontiers in microbiology 2022, 13, 963917.

Liu, B.; Zhu, X.; Cui, Y.; Wang, W.; Liu, H.; Li, Z.; Guo, Z.; Ma, S.; Li, D.; Wang, C.; Shi, Y. Consumption of dietary fiber from different sources during pregnancy alters sow gut microbiota and improves performance and reduces inflammation in sows and piglets. mSystems 2021, 26, 6

Xiao, H.H.; Lu, L.; Poon, C.C.; Chan, C.O.; Wang, L.J.; Zhu, Y.X.; Zhou, L.P.; Cao, S.; Yu, W.X.; Wong, K.Y., et al. The lignan-rich fraction from sambucus williamsii hance ameliorates dyslipidemia and insulin resistance and modulates gut microbiota composition in ovariectomized rats. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 2021, 137, 111372.

26. Line 289 change complicated to complex.

RE: Thank you for your reminding, it has been revised. Line 298

27. Line 291-292 Change: The data between the two types of samples analyzed by Adonis/PERMANOVA showed statistical significance. 

RE: Thank you for the advice, it has been revised. Line 300-301

28. What were the two types of samples? More detail about this citation is required.

RE: Thank you for the suggestion. In present study, “the two types of samples” mean “high and low apparent total tract digestibility(H- and L-AD) groups ”, maybe it is not apply. So we have changed it to “H- and L-AD groups”, and similar problems has been revised all over the manuscript . Line 300

29. Line 297 shannon and simpson -initial letters are capital

RE: Thank you for the suggestion, they have been revised. Line 305-306

30. Line 300  a link to the internet is found here :microbialcommunitystructure. Eliminate and correct

RE: Thank you for the advice, it has been revised. Line 322

31. Line 310-311 It is plausible to use 29 or 23 genera as biomarkers? This has to be discussed realistically.

RE: Thank you for your important suggestion, we have rewrite this section. Please see Result (Line209-229) and Discussion (Line320-357).

32. Line 319 Marques: also here there is a link. Eliminate it and add et al

RE: Thank you for the advice, it has been revised. Line367

33. Line 321-329. Just describing the data of other studies without proper discussion is not acceptable. Rewrite adding substantial analysis.

RE: Thank you for the comments which are very helpful to make this revision, and it has been rewrite. Please see Line326-357

Author Response File: Author Response.pdf

Reviewer 2 Report

Article Title: Identification of Gut Microbiota Affecting Fiber Digestibility in pig

This manuscript screens the gut microbiota related to fiber digestibility in pigs. Here the authors have identified the microbiota associated with apparent total tract digestibility of acid detergent fiber and neutral detergent fiber using suhuai breed. Here the experimental design seems to be quite simple, but the conclusions and the data interpretation done by the authors are of highly importance that demonstrates the phenotypic variations of fiber digestibility and further identify gut microbiota associated with ATTD, ADF and NDF.

Below are the comments related to the article and its content:

·         The article has been organised well by by providing an overview of the phenotypic variation between the ATTD, ADF and NDF.

·         The study size is really important in the gut microbiome studies, authors have thought this carefully and the considered the good sample size.

·         The differences of microbial community were demonstrated between high and low fiber digestibility groups.

·         A detailed description and the association of the potential pathways and its functions included.

·         I like the way authors have concluded the discussions.

·         The manuscript is well written and presented in a clear and well-structured manner. The figures, table and schemes in the main manuscript are clear and pertinent.

Saying that below are the few minor corrections that need to be addressed before accepting the article for publishing.

·         I was wondering why the short chain fatty acids were not discussed in the discussions, as they are very important in fiber digestion.

·         Figures are blurry, it would be better to increase the font size of the figures and choose the appropriate colours, where it will not affect the colour-blind readers.

·         Figure 3 is an important piece of this article but the demonstrations is not great, it would be best if authors consider other way of representation.

Author Response

Thank you for your valuable comments and kind consideration of revision. This manuscript has been revised considering all the comments raised from the reviewers. Any changes made based on the reviewers’ comments are highlighted with blue color fonts. The point-by-point responses to your and the reviewers’ comments are listed below.

 

1. I was wondering why the short chain fatty acids were not discussed in the discussions, as they are very important in fiber digestion.

RE: Thank you for the comments which are very helpful to make this revision. We completely agree with you. The discussion about the short chain fatty acids has been added. Line359-373

2. Figures are blurry, it would be better to increase the font size of the figures and choose the appropriate colours, where it will not affect the colour-blind readers.

RE: Thank you so much for the important reminds. Figure 1 and figure 3 have been revised. Line185, 230

3. Figure 3 is an important piece of this article but the demonstrations is not great, it would be best if authors consider other way of representation.

RE: Thank you for the thoughtful suggestion. It has been improved,  Linear discriminate analysis effect size (LEfSe) analysis has been changed to groups comparison analyses with Wilcoxon rank-sum test. Line230

Author Response File: Author Response.pdf