Relationship between true digestibility of dietary phosphorus and gastrointestinal bacteria of goats

Lizhi Wang; Ali Mujtaba Shah; Yuehui Liu; Lei Jin; Zhisheng Wang; Bai Xue; Quanhui Peng

doi:10.1371/journal.pone.0225018

Peer Review History

Original SubmissionOctober 24, 2019
20 Jan 2020 Decision Letter - Suzanne L. Ishaq, Editor PONE-D-19-29759 Relationship between True Digestibility of dietary Phosphorus and Gastrointestinal Bacteria of Goats PLOS ONE Dear Dr. Wang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. The reviewers were split on their opinion of the manuscript. In addition to a number of minor edits, there is a recommendation to strengthen and clarify the hypothesis and goals of the manuscript, all of which I agree with. I would like to give this opportunity to the authors to comment and perhaps revise their paper. If the authors choose to submit a revision, I may solicit the opinion of a third reviewer. We would appreciate receiving your revised manuscript by Mar 02 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Suzanne L. Ishaq, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. In your Methods section, please provide additional information on the animal research and ensure you have included details on : (1) methods of blood collection, e.g. volume, location from which samples were taken, methods of anesthesia if relevant (2) methods of goat sacrifice, and (3) efforts to alleviate suffering. 3. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability. Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized. Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access. We will update your Data Availability statement to reflect the information you provide in your cover letter. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The present aimed at characterizing the interrelation between true digestibility of phosphorus and gastrointestinal bacteria in goats. Therefore, the animals were kept at two different levels of P and at the end of the metabolic trials the animals were slaughtered and samples for characterizing the microbiome along the gastrointestinal axis were taken. Two major comments have to be guiven: 1. the differences in dietary P supply were marginal , i. e., according to plasma Ca and P concentrations the dietary treatment cannot be assigned as "LP". The whole study is not hypothesis driven. Thus, a clear concept is lacking. Reviewer #2: Line comment 28 phyla 49 , contributing to surface water eutrophication in locations where there is intensive monogastric livestock production [2,3]. Phytase is an enzyme that 67 technology has not been able to accomplish this work. Therefore, this study with 16S rRNA…. 69 true digestibilities of phosphorus 71 screening to identify high-efficiency…. 80 old healthy Nubian black goats with an average… 82 Goats were kept in individual pens with free access to water. 84 ) as shown in (Table 1) fed two times… 86 delete sentence starting with The formulas 90 Vitamin A, Vitamin D and Vitamin E is conventional nomenclature 97 collected. Was urine collected into acid? Or just mixed with acid after sampling? 97 An aliquot, representing 10% of the daily output was collected each day, mixed with 10 ml of 10% hydrochloric acid and composited. 100 6-day metabolism 103 jejunum…) were opened and digesta mixed and collected into tubes: rumen (8 tubes of 50 ml) …. 105 Ruminal sample pH was measured (PHS…. 107]. Ruminal fluid was strained through four layers of cheese cloth and 20 ml were mixed with 4 mL of 25%? Meta-phosphoric acid… 110 Samples from the GIT segments were stored at -80oC for further analysis. 116 gas chromatography 119 ground to pass a 40-mesh sieve 122 measure organic matter……crude ash. Neutral detergent fiber…What about mentioning specific procedure from AOAC book? 124 without sodium sulfite and corrected for residual ash. 136 true digestibility 142 TDP of all goats were calculated with mean… 142 Animals were grouped into high digestibility of phosphorus…. 147 and sterile phosphate buffered saline (pH 7) was used to rinse the liquid through 4 layers of cheesecloth and collected into a sterile tube (EP). The resulting liquid was immediately centrifuged at 10,000 x g and the supernatant gently removed. 152 used for DNA extraction from samples of ruminal and homogenized digesta from other organs following the manufacturers instructions using the procedure described in [19] 154 Agarose electrophoresis and NanoDrop 155 measure DNA quality of the samples. 184 SILVA 204 The TDP of the goats varied from 68.4% to 90.3% with an average of 83.3% (± 6.8%). 205 delete sentence. 206 The TDP of the 8 animals in the HP group 89.5% ± 2.9%) was significantly higher (than for the LP group (78.4% ± 1.21%). 209 was significantly higher (P<.05) than the LP group 212 not signficantly different (P>0.05) 230-359 While I recognize some phyla and genus, I am not familiar enough with literature to give a critical review. Yes, I have seen these in other research, but not knowledgeable enough to evaluate the research. 368 what is the feeding standard recommendation? 379 goats varied greatly. 382 but prior to our research, there was no published report on the variation of TDP… 383 earlier research showed that individual variations in feed efficiency of ruminants are common {… 393 microbial digestion is an important component of digestion in the 404 yet been reported. 405 the binding of free phosphorus with some substance in feed such as protein… 421 nutrient availability 445 CABI 451 feeding 504 excretion in Why not in figure 1B and 3a CHANGE SYMBOLS. Use a solid square for HP rumen and an open square for LP rumen. Use a solid triangle for the abomasum HP and an open triangle for LP. Use a solid and open circle for jejunum and open and closed diamond for the colon. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Steve Hart [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225018.r001
Revision 1
6 Feb 2020 Author Response Editor: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Response:We download the PLOS ONE style templates from the website, and formatted our manuscript as requested. Editor: In your Methods section, please provide additional information on the animal research and ensure you have included details on: (1) methods of blood collection, e.g. volume, location from which samples were taken, methods of anesthesia if relevant (2) methods of goat sacrifice, and (3) efforts to alleviate suffering. Response: we have revised our manuscript and the related contents were presented in L105-112 as “The next day after the end of the metabolism experiment, 10 ml of blood was collected from the jugular vein of each goat using a disposable vacuum blood collection tube before feeding in the morning. After anticoagulation, the plasma was collected and stored at -20 °C until use. Then the goats were slaughtered. Pre-harvest handling was conducted in accordance with good animal welfare practices, and slaughtering procedures followed actual chinese law on animal production and sanitary inspection.” Editor: In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. Response: In the “Data Availability Statement”, we state that “All the data of sequence in current research were placed in NCBI's sequence read archive (SRA) database with the accession No. SRP185613.” Editor: PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. Response: The ORCID iD for Lizhi Wang is :https://orcid.org/0000-0003-3074-4273, which has been authenticated in Editorial Manager. point-by-point responses to the reviewers Responses to Reviewer1: Two major comments have to be given: Reviewer1: the differences in dietary P supply were marginal , i. e., according to plasma Ca and P concentrations the dietary treatment cannot be assigned as "LP". Response: We are so sorry, probably because our presentation is not clear enough, the reviewer did not fully understand the way of animal grouping. Please allow us to explain it here. In the present study, the “LP” means low true digestibility of phosphorus rather than low dietary P level. The true digestibility of dietary phosphorus was determined by a two-stage metabolic experiment according to the Difference Level Technique (DLT). The dietary levels of phosphorus in the first and second metabolic experiments were 2.2 and 3.3 g/kg, respectively. During the two metabolic experiments, all of the 28 goats were fed the same diet, and then their true digestibility of dietary phosphorus was calculated according to DLT. According to the true digestibility of dietary phosphorus, the 28 goats were grouped into HP, MP and LP group. Reviewer1: The whole study is not hypothesis driven. Thus, a clear concept is lacking. Response: In fact, the hypothesis driving our study is implicit in the introduction, except that it is not clearly stated. According to your comment, to highlight the hypothesis of this study, we revised the our manuscript in L70-73 as “Therefore, in this study, we hypothesized that the digestibility of feed phosphorus in ruminants was influenced by the structure and composition of their gastrointestinal microbiota, and the 16S rRNA high-throughput sequencing technology was used to compare the bacterial diversity in the GIT of goats with different true digestibility of phosphorus (TDP).”. Responses to Reviewer2: Reviewer 2: L28.phyla Response: We have revised it in L29. Reviewer 2: L49.paying to surface water eutrophication in parts where the livestock production of monogastric is intensive [2, 3] .Phytase a type of phosphatase enzyme that catalyzes…. Response: we have revised in L50 as “contributing to surface water eutrophication in locations where there is intensive monogastric livestock production [2, 3]. Phytase is an enzyme that…” Reviewer 2: L67.technology has not been able to complete this work. In view of this fact, in this study the 16S rRNA…. Response:we have revised in L69 as“Therefore, in this study the 16S rRNA….” Reviewer 2: L69.true digestibility of phosphorus (TDP)…. Response:we have revised in L67 as “true digestibilities of phosphorus….’’ Reviewer 2: L71. screening to identify high-efficiency. Response:we have revised in L71 as “screening to identify high-efficiency….” Reviewer 2: L80. old healthy Nubian black goats with an average…. Response:we have revised in L80 as“old female healthy Nubian black goats with an average…” Reviewer 2: L82.All experimental goats were kept in pens (individual) with free choice to water. Response:we have revised in L82 as “Goats were kept in individual pens with free access to water”. Reviewer 2: L84. …presented in (Table 1) at two times at 8:00 am and 5:00 pm. Response:we have revised in L84 as “as shown in (Table 1) fed two times…” Reviewer 2: L86. delete sentence starting with The formulas Response: we have delete sentence starting with the formulas. Reviewer 2: L90.VA: 2 937 IU, VD: 343 IU, VE: 30 IU. Response: we have revised in L90 as“Vitamin A, Vitamin D and Vitamin E” Reviewer 2: L97 collected. Was urine collected into acid? Or just mixed with acid after sampling? Response: During the metabolism trial period, all the urine and feces were collected each day, and the feces and urine were sampled. The samples were mixed with hydrochloride. Reviewer 2: L100.the 6-days metabolism Response: we have revised in L100 as“6-day metabolism” Reviewer 2: L103.the GIT organs (rumen, abomasum, jeujinu, cecum and colon) were unglued to collect rumen (8 tubes, each of 50 mL), abomasums (4 tubes, each 15 mL), jejunum (4 tubes, each 15 mL), cecum (4 tubes, each 15 mL) and colon (4 tubes, each 15 mL) content . Response: we have revised inL103 as“jejunum…) were opened and the digesta was mixed and collected into tubes: rumen (8 tubes of 50 ml) ….” Reviewer 2: L105. A tube of ruminal sample was subjected to measure the pH (PHS… Response: we have revised in L105 as“Ruminal sample pH was measured (PHS….” Reviewer 2: L107. Ruminal fluid was strained through four layers of cheese cloth and 20 ml were mixed with 4 mL of 25%? Meta-phosphoric acid… 110 Samples from the GIT segments were stored at -80oC for further analysis. Response:We have revised in L117 as “Subsequently, this rumen fluid (approximately 20 mL) was mixed with 5mL solution of 25% metaphosphoric acid and preserved at -20°C for further calculation of the volatile fatty acid concentration (VFA; acetate, propionate, and butyrate).” Reviewer 2: L110.The other remnants of GIT segment samples were stored at -80°C for further assessment. Response: we have revised in L110 as“Samples from the GIT segments were stored at -80℃ for further analysis.” Reviewer 2: L116…. gas chromatography Response: we have revised in L116 as“gas chromatography” Reviewer 2: L119. ground to pass a 40-mesh sieve Response: we have revised in L119 as“ground to pass a 40-mesh sieve” Reviewer 2: L122. measure organic matter…crude ash. Neutral detergent fiber…What about mentioning specific procedure from AOAC book? Response: we have revised in L134-135 as “Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were measured according to the procedures of AOAC (method NO. 973.18 C, 1990).” Reviewer 2: L124…..with the filter bag technique lacking of sodium sulfite and articulated with ash residual. Response: we have revised in L134-138. Reviewer 2: L136. True digestibility Response: we have revised in L136 as “true digestibility” Reviewer 2: L142. TDP of all goats were calculated with mean… Response: we have revised in L142 as “TDP of all goats were calculated with mean…” Reviewer 2: L142. Animals were grouped into high digestibility of phosphorus…. Response: we have revised in L142 as“Animals were grouped into high digestibility of phosphorus….” Reviewer 2: L147. and sterile phosphate buffered saline (pH 7) was used to rinse the liquid through 4 layers of cheesecloth and collected into a sterile tube (EP). The resulting liquid was immediately centrifuged at 10,000 x g and the supernatant gently removed. Response: we have revised in L147 as“and sterile phosphate buffered saline (pH 7) was used to rinse the liquid through 4 layers of cheesecloth and collected into a sterile tube (EP). The resulting liquid was immediately centrifuged at 10,000 x g and the supernatant gently removed.” Reviewer 2: L152. used for DNA extraction from samples of ruminal and homogenized digesta from other organs following the manufacturers instructions using the procedure described in [19] Response: we have revised in L152 as “used for DNA extraction from samples of ruminal and homogenized digesta from other organs following the manufacturers instructions using the procedure described in [19]” Reviewer 2: L154. agrose electrophoresis and NanDrop 8000 spectrophotometer…. Response: we have revised in L154 as “Agarose electrophoresis and NanoDrop….” Reviewer 2: L155….. measure DNA quality of the samples. Response: we have revised in L155 as “measure DNA quality of the samples.” Reviewer 2: L184…..SILVA database…. Response: we have revised in L184 as “SILVA” Reviewer 2: L204. The TDP of the 28 goats highly varied (from 68.38% to 90.26%), and the average was 83.28% ± 6.81%. Response: we have revised in L204 as “The TDP of the goats varied from 68.4% to 90.3% with an average of 83.3% (± 6.8%).” Reviewer 2: L205. The TDP of the 8 animals in the HP group 89.5% ± 2.9%) was significantly higher (than for the LP group (78.4% ± 1.21%). Response: we have revised in L229-230 as “The TDP of the 8 animals in the HP group (89.5% ± 2.9%) was significantly higher than the LP group (78.4% ± 1.21%).” Reviewer 2: L209. was significantly higher (P<0.05) than the LP group),…. Response: we have revised in L209 as“was significantly higher (P<0.05) than the LP group” Reviewer 2: L209….. not significantly different (P>0.05)…. Response: we have revised in L209 as “not significantly different (P>0.05)” Reviewer 2: L230-359 While I recognize some phyla and genus, I am not familiar enough with literature to give a critical review. Yes, I have seen these in other research, but not knowledgeable enough to evaluate the research. Response: thank you. Reviewer 2: L368.what is the feeding standard recommendation? Response: The recommended phosphorus level of the Chinese meat sheep and goat feeding standard (NY/T 816-2004) is 4.0-5.0 g/kg DMI. In the present study, the phosphorus dietary levels in the first and second metabolic experiments were 2.2 and 3.3 g/kg, respectively. We have revised in L394 as “…Chinese meat sheep and goat feeding standard (4.0-5.0 g/kg DMI, NY/T 816-2004)”. Reviewer 2: L379. …. goats varied greatly …. Response: we have revised in L379 as “goats varied greatly.” Reviewer 2: L382…but prior to our research, there was no published report on the variation of TDP…. Response: we have revised in L382 as “but prior to our research, there was no published report on the variation of TDP…” Reviewer 2: L383…. earlier research showed that individual variations in feed efficiency of ruminants are common [35-37]. Response: we have revised in L383 as “earlier research showed that individual variations in feed efficiency of ruminants are common […” Reviewer 2: L393. microbial digestion is an important component of digestion in the… Response: we have revised in L393 as “microbial digestion is an important component of digestion in the…” Reviewer 2: L404.…. yet been reported. Response: we have revised in L404 as “yet been reported.” Reviewer 2: L405…. the binding of free phosphorus with some substance in feed such as protein…… Response: we have revised in L405 as “the binding of free phosphorus with some substance in feed such as protein…” Reviewer 2: L421…nutrient availability… Response: we have revised in L421 as “nutrient availability” Reviewer 2: 445 CABI Response: we have revised in L485 as “CABI” Reviewer 2: 451 feeding Response: we have revised in L491 as “feeding”. Reviewer 2: 504 excretion in Response: we have revised in L540 as “excretion in ruminants” Reviewer 2: Why not in figure 1B and 3a CHANGE SYMBOLS. Use a solid square for HP rumen and an open square for LP rumen. Use a solid triangle for the abomasum HP and an open triangle for LP. Use a solid and open circle for jejunum and open and closed diamond for the colon. Response: Sorry, we can't revise our article as you suggested. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0225018.r002
2 Mar 2020 Decision Letter - Suzanne L. Ishaq, Editor PONE-D-19-29759R1 Relationship between True Digestibility of dietary Phosphorus and Gastrointestinal Bacteria of Goats PLOS ONE Dear Dr. Wang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Both reviewers felt that the authors had made substantial improvements to the manuscript, but were mixed on the manuscript's readiness for publication. Reviewer 2 suggested a few minor spelling corrections, and Reviewer 1 felt that the authors had not sufficiently addressed one of their previous concerns on the interpretation. If the authors address this comment in the manuscript, it may require substantial changes to their discussion. If they chose not to, the authors should provide ample justification in their response as to why they felt they had already addressed this. I encourage you to consider these additional changes, and to submit a revised manuscript. We would appreciate receiving your revised manuscript by Apr 13 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Suzanne L. Ishaq, PhD Academic Editor PLOS ONE Journal Requirements: In your Methods section, please provide methods of animal sacrifice and methods of anesthesia and/or analgesia. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: (No Response) Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: (No Response) Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: (No Response) Reviewer #2: No ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Despite the authors have considered most comments originating from the first review process there still remains an open question. The authors claim some kind of interrelation between true phosphorus digestibility and the bacterial community. Based on this assumption quantitative differences in microbial protein synthesis could also be taken into account. Since the N:P ratio in microbial cell mass is rather constant differences in microbial protein synthesis would result in changes of true phosphorus digestibility. This should at least be taken into consideration in the discussion of the manuscript. Reviewer #2: Still a few errors to correct to polish MS 99 10% sample of each samping liquid is not random 111 13}. Rumen samples were filtered through four layers of cheesecloth into 3 tubes. 120 and gas chromatography 123 ground to pass 217 were not significantly 375 as compared to pigs ( ) or chickens ( ). ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Steve Hart [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225018.r003
Revision 2
16 Mar 2020 Author Response Point-by-point Responses We greatly appreciate the insightful and careful review by the editor and reviewers and have made the following revisions. Responses to Reviewer1 Reviewer1: Despite the authors have considered most comments originating from the first review process there still remains an open question. The authors claim some kind of interrelation between true phosphorus digestibility and the bacterial community. Based on this assumption，quantitative differences in microbial protein synthesis could also be taken into account. Since the N:P ratio in microbial cell mass is rather constant，differences in microbial protein synthesis would result in changes of true phosphorus digestibility. This should at least be taken into consideration in the discussion of the manuscript. Response: We have tried our best to revise our article according to the opinion of the reviewer. We agree with the reviewer that the N:P ratio in microbial cell mass is rather constant. However, the reference about “the differences in microbial protein synthesis would result in changes of true phosphorus digestibility” is not much. At last, we revise our manuscript in L432-440 as “Besides being relate to the ability of rumen microorganisms to produce phytase, the correlation between gastrointestinal bacteria and TDP in goats found in this study may also be related to the difference in rumen microbial protein synthesis between groups. Previous research found that the ratio of nitrogen to phosphorus in ruminal microbial cell mass is rather constant [49]，differences in microbial protein synthesis would result in changes of true phosphorus digestibility. Unfortunately, this study did not measure the activity of microbial phytase in the GIT and the amount of microbial protein synthesis in the rumen. Therefore, it was uncertain that the improvement of feed phosphorus digestibility was achieved through the increases of microbial phytase secretion or microbial protein synthesis.” Responses to Reviewer2: Reviewer 2: 99 10% sample of each samping liquid is not random Response: According to your comment, we revise our manuscript in L98-100 as “During the metabolism trial period all the urine and feces were collected. For nitrogen fixation, the 10% of each urine and feces samples were collected each day and then mixed with 10ml of hydrochloride (10%, v/v).” Reviewer 2: 111 13}. Rumen samples were filtered through four layers of cheesecloth into 3 tubes. Response: According to your comment, we revise our manuscript in L112-113 as “Rumen samples were filtered through four layers of cheesecloth into 3 tubes.” Reviewer 2: 120 and gas chromatography Response: According to your comment, we revise our manuscript in L120 as “and gas chromatography”. Reviewer 2: 123 ground to pass Response: According to your comment, we revise our manuscript in L 123 as “ground to pass”. Reviewer 2: 217 were not significantly Response: According to your comment, we revise our manuscript in L217 as “were not significantly”. Reviewer 2:375 as compared to pigs ( ) or chickens ( ). Response: According to your comment, we revise our manuscript in L 375 as “which was higher as compared to pigs [31] or chickens [32].”. https://doi.org/10.1371/journal.pone.0225018.r004
27 Mar 2020 Decision Letter - Suzanne L. Ishaq, Editor PONE-D-19-29759R2 Relationship between True Digestibility of dietary Phosphorus and Gastrointestinal Bacteria of Goats PLOS ONE Dear Dr. Wang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. I would to thank the authors for their attentiveness in addressing reviewer comments. Both reviewers are satisfied with the changes that have been made, and I agree that the manuscript is acceptable for publication. One reviewer noted several grammatical things to correct, and these were numerous enough that I felt it would be easier to correct these as a new submission rather than during the proofing process. Please make the recommended grammatical changes, and I will accept the resubmission without sending it for additional review. In addition, to comply with PLOS ONE's policy on animal research (https://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research), please provide methods of animal sacrifice and/or methods of anesthesia and/or analgesia in your Methods section. We would appreciate receiving your revised manuscript by May 10 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Suzanne L. Ishaq, PhD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: No ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: All comments have been adressed adequately and therefore, this manuscript is now in a version to contribute to new findings regarding P homeostasis in ruminants. Reviewer #2: There are a few changes to polish the MS. Line correction 21 Relationship instead of connection 26 Delete And aBacterial 16S rNA gene amplicons were sequenced from the rumen, abomasum, jejunum, cecum and Colten contents of the goats. 66 accomplish this instead of complete this 94 stages 141 true 233 A total of 5,711,204 high quality sequences were produced by the 16S rNA analysis, 915,084 in the rumen, 931,464 in the abomasum, 900,688 in the jejunum, 909,122 in the colon and 813,476 in the cecum. 236 in the rumen, the abomasum, the jejunum etc. 286 top 10 most plentiful 324 significantly 325 significantly compared 326 significantly 330 significantly 331 compared 337 in relative 339 significantly significantly 341 significantly 342 as compared 342 significantly 433 The correlation between TDP and microbial genus may not only be caused by the ability of the microbial genus to produce phytase, but may also be related to the difference…. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Steve Hart [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225018.r005
Revision 3
28 Mar 2020 Author Response Point-by-point Responses We greatly appreciate the insightful and careful review by the editor and reviewers and have made the following revisions. Responses to editor: Editor: In addition, to comply with PLOS ONE's policy on animal research (https://journals.plos.org/plosone/s/submission-guidelines#loc-animal-research), please provide methods of animal sacrifice and/or methods of anesthesia and/or analgesia in your Methods section. Response: We revised our MS as requested in L75-78 Materials and methods section as “The research procedure (Protocol Number: 2018-12825-01) used in current study was approved by Animals policy and welfare committee of Agricultural research organization of Sichuan province China and in agreement with rules of the Animal Care and Ethical Committee of the Sichuan Agricultural University (SAU).”, and in L108-110 as “Then the goats were slaughtered. Pre-harvest handling was conducted in accordance with good animal welfare practices, and slaughtering procedures followed actual Chinese law on animal production and sanitary inspection.” Responses to Reviewer2: Reviewer #2: There are a few changes to polish the MS. Reviewer 2: 21 Relationship instead of connection Response: According to your comment, we revise our manuscript in L21 as “relationship….” Reviewer 2: 26 Delete And aBacterial 16S rNA gene amplicons were sequenced from the rumen, abomasum, jejunum, cecum and Colten contents of the goats Response: According to your comment, we revise our manuscript in L26 as “The bacterial 16S rRNA gene amplicons were sequenced from the rumen, abomasum, jejunum, cecum and colon contents of the goats.” Reviewer 2: 66 accomplish this instead of complete this Response: According to your comment, we revise our manuscript in L66 as “accomplish …” Reviewer 2: 94 stages Response: According to your comment, we revise our manuscript in L94 as “ stages…” Reviewer 2: 141 true Response: According to your comment, we revise our manuscript in L141 as “ true….” Reviewer 2: 233 A total of 5,711,204 high quality sequences were produced by the 16S rNA analysis, 915,084 in the rumen, 931,464 in the abomasum, 900,688 in the jejunum, 909,122 in the colon and 813,476 in the cecum. Response: According to your comment, we revise our manuscript in L233 as “A total of 5,711,204 high quality sequences were produced by the 16S rNA analysis, 915,084 in the rumen, 931,464 in the abomasum, 900,688 in the jejunum, 909,122 in the colon and 813,476 in the cecum.” Reviewer 2: 236 in the rumen, the abomasum, the jejunum etc. Response: According to your comment, we revise our manuscript in L236 as “were recognized in the rumen, in the abomasum 5,810, in the jejunum 5,611, in the colon 8,886 and in the cecum……” Reviewer 2: 286 top 10 most plentiful Response: According to your comment, we revise our manuscript in L286 as “top 10 most plentiful…..” Reviewer 2: 324 significantly Response: According to your comment, we revise our manuscript. Reviewer 2: 325 significantly compared Response: According to your comment, we revise our manuscript in L325 as “was significantly (p<0.05) lower in the LP compared to HP group……” Reviewer 2: 326 significantly Response: According to your comment, we revise our manuscript in L326. Reviewer 2: 330 significantly Response: According to your comment, we revise our manuscript in L330. Reviewer 2: 331 compared Response: According to your comment, we revise our manuscript in L331. Reviewer 2: 337 in relative Response: According to your comment, we revise our manuscript in L337 as “in relative..” Reviewer 2: 339 significantly significantly Response: According to your comment, we revise our manuscript in L339. Reviewer 2: 341 significantly Response: According to your comment, we revise our manuscript in L341. Reviewer 2: 342 as compared Response: According to your comment, we revise our manuscript in L342. Reviewer 2: 342 significantly Response: According to your comment, we revise our manuscript in L342. Reviewer 2: 433 The correlation between TDP and microbial genus may not only be caused by the ability of the microbial genus to produce phytase, but may also be related to the difference…. Response: According to your comment, we revise our manuscript in L433 as “The correlation between TDP and microbial genus may not only be caused by the ability of the microbial genus to produce phytase, but may also be related to the difference….” https://doi.org/10.1371/journal.pone.0225018.r006
29 Apr 2020 Decision Letter - Suzanne L. Ishaq, Editor PONE-D-19-29759R3 Relationship between True Digestibility of dietary Phosphorus and Gastrointestinal Bacteria of Goats PLOS ONE Dear Dr. Wang, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that clarifies the following information: -how and where the goats were slaughtered -the method of anaesthesia or analgesia used, if relevant Please address these in the methods section, as these revisions were previously requested and are required to move forward with publication. We would appreciate receiving your revised manuscript by Jun 13 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Suzanne L. Ishaq, PhD Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0225018.r007
Revision 4
3 May 2020 Author Response Comments: -how and where the goats were slaughtered -the method of anaesthesia or analgesia used, if relevant Response: We have revised as per suggestion thanks. The goats were slaughtered at slaughter house in Teaching and Research Base, Animal Nutrition Institute, Sichuan Agricultural University. All the goats were slaughter according to the American Veterinary Medical Association (AVMA) guidelines for the humane slaughter of animals. Note: (The animals were stunned by captive bolt and the exsanguination from the jugular vein was carried out) Attachments Attachment Submitted filename: Response to reviewers -.docx https://doi.org/10.1371/journal.pone.0225018.r008
6 May 2020 Decision Letter - Suzanne L. Ishaq, Editor Relationship between True Digestibility of dietary Phosphorus and Gastrointestinal Bacteria of Goats PONE-D-19-29759R4 Dear Dr. Wang, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Thank you for supplying that additional detail to the methods section. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Suzanne L. Ishaq, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0225018.r009
Formally Accepted
8 May 2020 Acceptance Letter - Suzanne L. Ishaq, Editor PONE-D-19-29759R4 Relationship between True Digestibility of dietary Phosphorus and Gastrointestinal Bacteria of Goats Dear Dr. Wang: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Suzanne L. Ishaq Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0225018.r010

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .