Calcium Propionate Supplementation Mitigated Adverse Effects of Incubation Temperature Shift on In Vitro Fermentation by Modulating Microbial Composition
, Xilin Wang 1,2, Shenfei Long 1,2, Jiangong Li 1,2, Zhenlong Wu 1,2, Yao Guo 1,2, Fang Sun 3 and Zhaohui Chen 1,2,*
Round 1
Reviewer 1 Report
The aim of the study submitted for evaluation was to determine the effect of CaP addition and changes in incubation temperature on rumen in vitro fermentation parameters, gas production, and microbial communities.
The methodological scope of the work, the content of subsequent chapters and conclusions do not raise any major comments or objections.
A few minor comments, rather of an editorial nature:
The order of the keywords.
The order of the subsections in the results section should be changed as follows:
3.1. Fermentation parameters
3.2. Gas production
3.2.1. Cumulative gas production
3.2.2. CH4 and CO2 production
Unify the way of citation, in the introduction:
Line 43: there is Graham et al. missing 8 in brackets, not at the end of the sentence.
Line 45: Kennedy and Milligan (1978), not the year but the 9 which is at the end of the sentence.
Line 47, 54 i 57: similarly.
Line 64: [13] or (Bergman, 1974), similarly in line 69: [15] or (Schulman et al., 1976).
There is no need to repeat the titles of charts or tables in the text, e.g. in line 192-193: “The results of gas production affected by incubation temperature conditions and CaP supplementation are shown in Figure 1 and Figure 2”, it is enough to refer them and give in the next sentence in brackets.
In conclusion, it was stated that: “the addition of 2.5% CaP could alleviate the adverse effect of ITS on in vitro fermentation parameters and maintain the reduction of CH4 production by adjusting the microbial composition”, which is not entirely consistent with the results of other studies e.g: Zhang F. et al. (44), which suggest that supplementation with calcium propionate improves fermentation and bacterial diversity in the rumen, but to a small extent determines the composition of the major bacterial communities in the rumen of early lactating dairy cows. However, the authors refer to these results in the discussion, pointing to differences in in vivo and in vitro fermentation.
The comments presented do not affect the final positive evaluation of the work. These are, of course, only suggestions for possible use by the authors. With minor changes, the work should be published in the Fermentation journal.
Author Response
Response to Reviewer 1 Comments
Dear reviewer: Thank you very much for your valuable and helpful comments. Those comments are good for us to improve our paper further. We have studied comments carefully and revised the manuscript thoroughly according to comments, and the amendments are highlighted in red in the revised manuscript. We appreciate the warm work from you, and hope that the corrections will meet with approval. Once again, thank you for your helpful comments and suggestions. And we look forward to hearing from you about our revised paper. Kind regards!
Dr. Zhaohui Chen
State Key Laboratory of Animal Nutrition
Ministry of Agriculture and Rural Affairs Feed Industry Centre
College of Animal Science and Technology
China Agricultural University
2023-05-21
Comment 1: The order of the keywords.
Response 1: Thank you for your suggestion. We have revised the order of the keywords in accordance with the key research content of our manuscript in line 32-33. The current order of keywords is "In vitro fermentation; Incubation temperature; Microbial abundance; Calcium propionate; Methane production".
Comment 2: The order of the subsections in the results section should be changed as follows:
3.1. Fermentation parameters
3.2. Gas production
3.2.1. Cumulative gas production
3.2.2. CH4 and CO2 production
Response 2: Thank you for your valuable comment. We have changed the order of subsections based on your suggestion (Lines 186, 203, 204 and 228).
Comment 3: Unify the way of citation, in the introduction:
Line 43: there is Graham et al. missing 8 in brackets, not at the end of the sentence.
Line 45: Kennedy and Milligan (1978), not the year but the 9 which is at the end of the sentence.
Line 47, 54 i 57: similarly.
Line 64: [13] or (Bergman, 1974), similarly in line 69: [15] or (Schulman et al., 1976).
Response 3: Thanks for your comment. we have revised the manuscript based on your suggestions to ensure that all citations follow the correct style on lines 43, 46, 47 and 53 and 55. Besides, according to the comment of reviewer 2, we have removed old references such as Bergman (1974) and Schulman et al. (1976) and replacing them with more recent publications that are relevant to the topic.
Comment 4: There is no need to repeat the titles of charts or tables in the text, e.g. in line 192-193: “The results of gas production affected by incubation temperature conditions and CaP supplementation are shown in Figure 1 and Figure 2”, it is enough to refer them and give in the next sentence in brackets.
Response 4: Thank you for your valuable comment. We have revised the text accordingly and removed the repetition of the titles of charts or tables. For example, in line 187-188, I stated the results directly and added (Table 2) at the end of the sentence. And please see lines 205-206, 229-231. 243-244 and 263-266 for the modification of other parts
Comment 5: In conclusion, it was stated that: “the addition of 2.5% CaP could alleviate the adverse effect of ITS on in vitro fermentation parameters and maintain the reduction of CH4 production by adjusting the microbial composition”, which is not entirely consistent with the results of other studies e.g: Zhang F. et al. (44), which suggest that supplementation with calcium propionate improves fermentation and bacterial diversity in the rumen, but to a small extent determines the composition of the major bacterial communities in the rumen of early lactating dairy cows. However, the authors refer to these results in the discussion, pointing to differences in in vivo and in vitro fermentation.
The comments presented do not affect the final positive evaluation of the work. These are, of course, only suggestions for possible use by the authors. With minor changes, the work should be published in the Fermentation journal.
Response 5: Thank you for your evaluation on our manuscript, We have discussed the results of Zhang et al. [44] in the discussion section and pointed out the differences between our study and theirs, particularly regarding in vivo and in vitro fermentation (Line 332-335). In addition, we have also acknowledged the limitations of our study and point that the need for further research to fully understand the effects of CaP supplementation on in vivo rumen fermentation and microbial composition (Lines 418-421).
Reviewer 2 Report
1. There is no well-founded hypothesis that drinking water decreases rumen temperature. The authors only assume that it drops to 30 degrees Celsius, and this value is not very well founded. Furthermore, why was only one In vitro fermentation run carried out? The most recommended is to perform three runs to reduce the error and have more repetitions and degrees of freedom.
2. The authors do not explain in detail in the experiment what the "temperature shift periodically" means, its determination value is very uncertain.
3. In the statistical analysis, it is best to also analyze the effect without and with calcium propionate
4. The Menke technique has been improved and the use of syringes to capture the gas is imprecise, there is a lot of loss. The authors do not report the degradability of dry matter. Gas production must be reported based on grams of dry matter or organic matter. It is not adequate to only report total gas.
5. The authors propose the analysis with the Orskov and McDonald equation, but they do not report results of rumen kinetics with the formula.
Author Response
Response to Reviewer 2 Comments
Dear reviewer: Thank you very much for your valuable and helpful comments. Those comments are good for us to improve our paper further. We have studied comments carefully and revised the manuscript thoroughly according to comments, and the amendments are highlighted in red in the revised manuscript. We appreciate the warm work from you, and hope that the corrections will meet with approval. Once again, thank you for your helpful comments and suggestions. And we look forward to hearing from you about our revised paper. Kind regards!
Dr. Zhaohui Chen
State Key Laboratory of Animal Nutrition
Ministry of Agriculture and Rural Affairs Feed Industry Centre
College of Animal Science and Technology
China Agricultural University
2023-05-21
Response to reviewer 2
Comment 1: There is no well-founded hypothesis that drinking water decreases rumen temperature. The authors only assume that it drops to 30 degrees Celsius, and this value is not very well founded. Furthermore, why was only one In vitro fermentation run carried out? The most recommended is to perform three runs to reduce the error and have more repetitions and degrees of freedom.
Response 1: Thank you for your valuable comments. We appreciate your concerns regarding the assumption of rumen temperature and the number of In vitro fermentation runs in our study.
We have added the reason that hypothesis the rumen temperature drops to 30 °C in the manuscript (Lines 68-75).
Regarding the number of in vitro fermentation runs, we acknowledge that performing multiple replicates in in vitro fermentation experiments is indeed recommended to reduce errors and increase the degrees of freedom. However, it is worth mentioning that we also referred to several published studies, such as Wu et al. (2021), Zheng et al. (2022), and Cañaveral-Martínez et al. (2023), where researchers conducted single runs in their in vitro fermentation experiment. In addition, rumen fluid in our experiment were collected from three Angus cattle (460 ± 48 kg) fitted with permanent ruminal fistulas and mixed eventually (Lines 104-106), which also can reduce errors. Therefore, we argued that only conducted one run with 6 replicates for each treatment also can be accepted as a scientific way. But, we appreciate your suggestion of incorporating multiple fermentation runs in future studies to further enhance the reliability and statistical rigor of the findings. These information about the experiment procedure was shown in lines 104-122.
Comment 2: The authors do not explain in detail in the experiment what the "temperature shift periodically" means, its determination value is very uncertain.
Response 2: Thank you for your comment. We have added the detail definition of “temperature shift periodicity” in lines 90-92. The definition of temperature shift periodicity is specific temperature values for the transitions, which was determined based on previous studies and existing literature, mainly taking into consideration the rumen temperature fluctuations after the consumption of cold water in winter (as explained in manuscript lines 68-75).
Comment 3: In the statistical analysis, it is best to also analyze the effect without and with calcium propionate
Response 3: Thank you for your comment. We conducted analyses to compare the effects with and without calcium propionate in the original manuscript. The information of the analysis method shown in our manuscript in lines 173-174.
Comment 4: The Menke technique has been improved and the use of syringes to capture the gas is imprecise, there is a lot of loss. The authors do not report the degradability of dry matter. Gas production must be reported based on grams of dry matter or organic matter. It is not adequate to only report total gas.
Response 4: Thank you for your valuable comments. Regarding the Menke technique, we acknowledge the advancements and improvements that have been made in gas production measurement. However, due to limitations in our experimental setup, we utilized the syringe method for gas collection. Our experiment referred to published studies (e.g., Wu et al., 2021) that have also used the syringe method for gas collection in the same experimental setups. Besides, we also took measures to minimize gas losses by ensuring proper sealing and standardization process of the syringes.
Regarding the reporting of gas production, we would like to clarify that all values in the gas production results have indeed been calculated on a per 0.2 g dry matter substrate basis. We have made revisions in the manuscript accordingly to present that the gas production results are presented based on per 0.2 g dry matter substrate. Specifically, the revised part can be found in lines 22, 137-139, 206-207, 213, 221, 283, and 307.
Comment 5: The authors propose the analysis with the Orskov and McDonald equation, but they do not report results of rumen kinetics with the formula.
Response 5: Thank you for your comment. We have added the rumen kinetics results according to the Orskov and McDonald equation in Figure 2, including the results of the asymptotic gas production per 0.2 g DM substrate and the rate of gas production per hour (Line 220).
Reviewer 3 Report
Review of Manuscript Fermentation-2402781
The aim of the manuscript was to evaluate the effect of adding calcium propionate under different temperatures on in vitro fermentation parameters. I have for the authors the following remarks:
Major comments
In abstract I would recommend to give some values for increments and decrements to understand the magnitude of the effects
The M&M was described with enough detail.
In conclusion I would expect some recommendations as well for further studies or how to apply the results in the practice
Minor comments
L17: What do you mean with 12 cycles? Clarify here
L17-19: Results are not surprising by reducing temperature. I do not understand what the application on practice could be
L18 &20: give in % the level of decrease/increase to understand the magnitude
L91: describe the CaP used
L196: What is the theoretical maximum gas production? I think this was not clarified in M&M. Is this a+b?
L192-198: I recommend highlighting some differences between treatments. The description of results done here are too general
L200: Abbreviations are confusing. Use ITS as in the document instead of TS
L216-220: The p-value must be here ≤0.02. Use please always the exact p-values. Use always the exact p-values that are reported in the tables
L236: give here the p-value for all
Author Response
Response to Reviewer 3 Comments
Dear reviewer: Thank you very much for your valuable and helpful comments. Those comments are good for us to improve our paper further. We have studied comments carefully and revised the manuscript thoroughly according to comments, and the amendments are highlighted in red in the revised manuscript. We appreciate the warm work from you, and hope that the corrections will meet with approval. Once again, thank you for your helpful comments and suggestions. And we look forward to hearing from you about our revised paper. Kind regards!
Dr. Zhaohui Chen
State Key Laboratory of Animal Nutrition
Ministry of Agriculture and Rural Affairs Feed Industry Centre
College of Animal Science and Technology
China Agricultural University
2023-05-21
Response to reviewer 3
The aim of the manuscript was to evaluate the effect of adding calcium propionate under different temperatures on in vitro fermentation parameters. I have for the authors the following remarks:
Major comments
Comment 1: In abstract I would recommend to give some values for increments and decrements to understand the magnitude of the effects
Response 1: Thank you for your comment. I have revised the abstract to include specific values for the increments and decrements (lines 20-23), providing a clearer understanding of the magnitude of the effects observed.
Comment 2: The M&M was described with enough detail.
Response 2: Thank you for your valuable feedback. In order to provide a clearer understanding of our research methods, we have made further revisions and additions to the M&M section (Lines 87-94, 113-114 and 137-139).
Comment 3: In conclusion I would expect some recommendations as well for further studies or how to apply the results in the practice
Response 3: Thank you for your valuable comments. In response, we have revised the conclusions section to provide recommendations in revised manuscript (Lines 418-421)
Minor comments
Comment 4: L17: What do you mean with 12 cycles? Clarify here
Response 4: Thank you for your comment. The term "12 cycles" refers to the total number of temperature transitions during the incubation period. Each cycle involves 4 hours of incubation at 39°C followed by an immediate transfer to 30°C for 2 hours. The term has been clarified in the revised manuscript (Line 17-18).
Comment 5: L17-19: Results are not surprising by reducing temperature. I do not understand what the application on practice could be
Response 5: Thank you for your comment. The application on practice of our findings lies in the application of adding 2.5% CaP to beef cattle rations in cold season. Our results demonstrate that dietary supplementation with 2.5% CaP has the potential to mitigate the negative effects of drinking cold water on ruminal fermentation and sustain the reduction in methane production by modulating microbial composition. These findings propose a practical strategy for enhancing rumen health and reducing methane emissions of beef cattle in winter. This point has been added to manuscripts (Lines 72-75 and Lines 418-421)
Comment 6: L18 &20: give in % the level of decrease/increase to understand the magnitude
Response 6: Thank you for your comment. We have provided the relevant percentage values (Line 20-23) in the manuscript to enhance the interpretation of the results.
Comment 7: L91: describe the CaP used
Response 7: Thank you for your comment. We have added the detailed information of the CaP in lines 88-89
Comment 8: L196: What is the theoretical maximum gas production? I think this was not clarified in M&M. Is this a+b?
Response 8: Thank you for your comment. We apologize for this confusion caused by the lack of clarification. We rewrote the expression about the gas production. Specifically, we have revised the “the theoretical maximum gas production” to “asymptotic gas production per 0.2 g DM substrate”, and rewrote the formula of the dynamic gas production parameters in lines 136-139, and the asymptotic gas production per 0.2 g DM substrate now is represented by b (Line 136).
Comment 9: L192-198: I recommend highlighting some differences between treatments. The description of results done here are too general
Response 9: Thank you for your valuable comment. we have revised this part to include more specific and detailed comparisons between treatments, highlighting the significant differences observed (Lines 205-210).
Comment 10: L200: Abbreviations are confusing. Use ITS as in the document instead of TS
Response 10: Thanks for your comment. We understand your suggestion to use "ITS" as in the document instead of "TS". However, we would like to clarify that "ITS" refers to the fixed effect of " Incubation temperature shift," while "TS" specifically represents a treatment group of " Temperature shift without CaP addition". These abbreviations have been used consistently throughout the manuscript to differentiate between different variables and treatment groups. Therefore, we prefer to keep useing "TS" in this context to ensure consistency and clarity in our presentation of the results.
Comment 11: L216-220: The p-value must be here ≤0.02. Use please always the exact p-values. Use always the exact p-values that are reported in the tables
Response 11: Thank you for your comment. We have reviewed our results and tables and added the exact p-values in the text for better clarity (Lines 190-192, 209-210 and 232-233).
Comment 12: L236: give here the p-value for all
Response 12: Thanks for your comment. We have added all the P-values here based on your suggestion (Lines 232-233).
Reviewer 4 Report
Dear authors, it seems to me that this is a well-written manuscript; however, its relevance in the scientific world is questionable because it is not economically viable considering that a low temperature significantly affects animal production.
Dear authors, it is necessary to describe it in the introduction because they decided to study lower temperatures.
Low temperatures decrease animal performance and are not beneficial for cattle. In that sense, why is your study interesting?
From what I understand, you put lower methane production above animal production. I think this is not a reasonable idea to stimulate a previously corroborated study.
Another question is: How low would the external temperature have to be for the rumen to reach 30 °C?
Line 37-38: Reference [4] and [5] are a reference of almost twenty years. In this sense, it is not correct to use the percentages cited because these data changed over time.
Lines 45-47: The reference used for this text is also very old. Also, many references are very old, it is necessary to update the references.
Table 1: Add the dry matter, ether extract, and lignin content of the diet.
Lines 192-193: Avoid using this type of expression because it does not add relevant information. Same comment for lines 215-216 and others throughout the text.
Results: The description of the results should be improved. Sometimes the description is difficult to read since it is sometimes controversial, this because the figure or table expresses one thought and the text another.
Lines 343-347: This is more of a description of results than a discussion.
Discussion: The discussion here is a great review; however, I am sorry for the lack of discussion of your own data.
Line 425: Describe ITS extensively here.
Author Response
Response to Reviewer 4 Comments
Dear reviewer: Thank you very much for your valuable and helpful comments. Those comments are good for us to improve our paper further. We have studied comments carefully and revised the manuscript thoroughly according to comments, and the amendments are highlighted in red in the revised manuscript. We appreciate the warm work from you, and hope that the corrections will meet with approval. Once again, thank you for your helpful comments and suggestions. And we look forward to hearing from you about our revised paper. Kind regards!
Dr. Zhaohui Chen
State Key Laboratory of Animal Nutrition
Ministry of Agriculture and Rural Affairs Feed Industry Centre
College of Animal Science and Technology
China Agricultural University
2023-05-21
Response to reviewer 4
Comment 1: Dear authors, it seems to me that this is a well-written manuscript; however, its relevance in the scientific world is questionable because it is not economically viable considering that a low temperature significantly affects animal production.
Response 1: Thank you for your valuable comments. As we noted in the introduction (line 49-51), we acknowledge that low temperature environment and direct drinking of cold water can have a significant impact on animal production. Our study suggests that dietary supplementation of 2.5% CaP may reduce the adverse effects of drinking cold water on rumen function and has economic value for production applications. I have supplemented and explained the purpose of this research in the induction (lines 57-59, 64-67 and 72-75) and the conclusion (lines 418-421).
Comment 2: Dear authors, it is necessary to describe it in the introduction because they decided to study lower temperatures.
Low temperatures decrease animal performance and are not beneficial for cattle. In that sense, why is your study interesting?
From what I understand, you put lower methane production above animal production. I think this is not a reasonable idea to stimulate a previously corroborated study.
Response 2: Thank you for your valuable suggestions. As I mentioned in response to comment 1, the objective of our study was to simulate the effects of cold water consumption during winter on rumen fermentation parameters and methane emissions, and investigate whether the addition of calcium propionate could mitigate the negative effects of rumen temperature reduction. We have added additional information in the induction (lines 57-59, 64-67 and 72-75) and the conclusion (lines 418-421) to better explain the rationale of our study. I hope this explanation is satisfactory and meets with your approval.
Comment 3: Another question is: How low would the external temperature have to be for the rumen to reach 30 °C?
Response 3: Thanks for your valuable comment. We have added the reason that hypothesis the rumen temperature drops to 30 °C in the induction (Lines 68-74)
Comment 4: Line 37-38: Reference [4] and [5] are a reference of almost twenty years. In this sense, it is not correct to use the percentages cited because these data changed over time.
Response 4: Thanks for your valuable comments, I have updated the references [4] and [5] to the 2022 and 2019 reference respectively, and changed the relevant content in this part of the introduction (Lines 39-40).
Comment 5: Lines 45-47: The reference used for this text is also very old. Also, many references are very old, it is necessary to update the references.
Response 5: Thank you for your suggestion. I have carefully reviewed the references in the manuscript and updated some of them to recent publications and changed the presentation accordingly in the text. Specifically, I updated references [8], [9], [13], [15], [24], [25], [46], and [47] to recent years.
Comment 6: Table 1: Add the dry matter, ether extract, and lignin content of the diet.
Response 6: Thanks for your comment, we have added the dry matter, ether extract, and lignin content of the diet in Table 1 (Line 98).
Comment 7: Lines 192-193: Avoid using this type of expression because it does not add relevant information. Same comment for lines 215-216 and others throughout the text.
Response 7: Thank you for your valuable comment. We have revised the expression type of the results part accordingly and removed the repetition of the titles of charts or tables. For example, in lines 187-188, we stated the results directly and added (Table 2) at the end of the sentence.
Comment 8: Results: The description of the results should be improved. Sometimes the description is difficult to read since it is sometimes controversial, this because the figure or table expresses one thought and the text another.
Response 8: Thank you for your valuable comments. We have carefully reviewed and revised the manuscript, such as lines 188-195, 205-210 and line 265, etc., to ensure that the description of the results accurately reflects the findings presented in the table and figure.
Comment 9: Lines 343-347: This is more of a description of results than a discussion.
Response 9: Thanks for your comment. We have revised and streamlined the presentation of this section. Please see lines 336 to 338 for specific modifications.
Comment 10: Discussion: The discussion here is a great review; however, I am sorry for the lack of discussion of your own data.
Response 10: Thank you for your valuable comments. We have made the necessary modifications to address this issue. Specifically, we have included a discussion of our own data in lines 313-317, 321-327, and 348-357 of the manuscript.
Comment 11: Line 425: Describe ITS extensively here.
Response 11: Thanks for your valuable suggestion, I have modified the ITS on lines 413 to 414 by changing "ITS" to " incubation temperature shift (39℃ for 4 h followed by 30℃ for 2 h, periodically)"
Round 2
Reviewer 2 Report
Authors made all corrections
Author Response
Response to Reviewer 2 Comments of Second Round
Dear reviewer: Thank you again for your valuable and helpful comments on our manuscript. Those comments are good for us to improve our paper further. We have studied comments carefully and revised the manuscript thoroughly according to comments, and the amendments are highlighted in red in the revised manuscript. We appreciate the warm work from you, and hope that the corrections will meet with approval. Once again, thank you for your helpful comments and suggestions. And we look forward to hearing from you about our revised paper. Kind regards!
Dr. Zhaohui Chen
State Key Laboratory of Animal Nutrition
College of Animal Science and Technology
China Agricultural University
2023-05-30
Response
Comments and Suggestions for Authors：Authors made all corrections
Response: We appreciate your kind approval of the revisions we made to our manuscript. We are grateful for your thorough review and valuable input, which have greatly contributed to improving the quality of our manuscript. Thank you for your support and guidance throughout the review process.
Reviewer 4 Report
Dear authors, now I can see the objective you are trying to achieve. Thank you very much for the improvement of the manuscript. However, I have a few more suggestions, because some changes are still needed. Now the reason for the manuscript and the problem it intends to solve is clearer.
Now you better describe why you studied temperature variation and the use of CaP. However, the introduction is disorganized/incomplete:
1. The addition of the new text changed the organization of the manuscript. Try to follow an organized sequence. Reorganized it.
2. The hypothesis is before the objective, in the same paragraph. Change the position.
3. In its current form, this hypothesis is an incomplete idea. Follow your objective as an example to improve/complete your hypothesis.
4. Lines 68-75, this is a good text, which helped me to understand why you studied the lower temperatures; however, it is incomplete. I think somewhere in the introduction, you need to complete this idea.
For example (Example only): X regions of the world have template climates with cold water temperatures (Z °C). When animals drink this low-temperature water, rumen fermentation is affected, more methane is produced, and animal performance is negatively affected. This was corroborated by ……….. On the other hand, calcium propionate showed potential to reduce the negative effects of lower water temperatures and improve rumen fermentation …………..
Lines 405-408: I don't understand this statement. How the absorption and metabolization of propionate in the rumen can maintain a constant concentration of propionate in the rumen?
Conclusion: This conclusion is more consistent with a results text than with a discussion text. You can use this text as "future implications" after the actual conclusion. To write the conclusion use the objective of the manuscript. Example (Example only): 30 °C rumen temperature affects rumen metabolism; however, X grams of calcium propionate lessens those effects and promotes an A% improvement in degradability of the diet. ….. [You text here (In summary ….), after the conclusion]
Author Response
Response to Reviewer 4 Comments of Second Round
Dear reviewer: Thank you again for your valuable and helpful comments on our manuscript. Those comments are good for us to improve our paper further. We have studied comments carefully and revised the manuscript thoroughly according to comments, and the amendments are highlighted in red in the revised manuscript. We appreciate the warm work from you, and hope that the corrections will meet with approval. Once again, thank you for your helpful comments and suggestions. And we look forward to hearing from you about our revised paper. Kind regards!
Dr. Zhaohui Chen
State Key Laboratory of Animal Nutrition
College of Animal Science and Technology
China Agricultural University
2023-05-30
Response
Dear authors, now I can see the objective you are trying to achieve. Thank you very much for the improvement of the manuscript. However, I have a few more suggestions, because some changes are still needed. Now the reason for the manuscript and the problem it intends to solve is clearer. Now you better describe why you studied temperature variation and the use of CaP. However, the introduction is disorganized/incomplete:
Comment 1: The addition of the new text changed the organization of the manuscript. Try to follow an organized sequence. Reorganized it. The hypothesis is before the objective, in the same paragraph. Change the position. Response 1: Thank you for your suggestion. We have reorganized the introduction as your recommendation. The hypothesis and objective are now presented in the same paragraph, with the hypothesis preceding the objective. (Lines 63-67)
Comment 2: In its current form, this hypothesis is an incomplete idea. Follow your objective as an example to improve/complete your hypothesis.
Response 2: Thank you for your comment. We have revised and improved the hypothesis to align it more effectively with the objective, as shown in lines 50-67.
Comment 3: Lines 68-75, this is a good text, which helped me to understand why you studied the lower temperatures; however, it is incomplete. I think somewhere in the introduction, you need to complete this idea.For example (Example only): X regions of the world have template climates with cold water temperatures (Z °C). When animals drink this low-temperature water, rumen fermentation is affected, more methane is produced, and animal performance is negatively affected. This was corroborated by ……….. On the other hand, calcium propionate showed potential to reduce the negative effects of lower water temperatures and improve rumen fermentation …………..
Response 3: Thank you for your comment. We have revised the introduction accordingly. In the revised version (lines 50-69), we have provided additional information about the regions characterized by cold water temperatures, emphasizing the potential of calcium propionate to mitigate the adverse effects associated with consuming lower water temperatures.
Comment 4: Lines 405-408: I don't understand this statement. How the absorption and metabolization of propionate in the rumen can maintain a constant concentration of propionate in the rumen?
Response 4: Thank you for your comment. We have provided a detailed explanation for the relatively constant absorption and maintenance of propionate concentration in the rumen after the exogenous addition of propionate. This explanation can be found in lines 388-399 of the revised manuscript.
Comment 5: Conclusion: This conclusion is more consistent with a results text than with a discussion text. You can use this text as "future implications" after the actual conclusion. To write the conclusion use the objective of the manuscript. Example (Example only): 30 °C rumen temperature affects rumen metabolism; however, X grams of calcium propionate lessens those effects and promotes an A% improvement in degradability of the diet. ….. [You text here (In summary ….), after the conclusion]
Response 5: We appreciate the reviewer's valuable comment and guidance regarding the revision of our conclusion. Based on the reviewer's suggestion, we have restructured the conclusion to align it more closely with the objective of the manuscript. We have also incorporated the suggested "future implications" section to provide further clarity on the potential applications of our findings (Lines 401-410). Thank you for your valuable input, which has contributed to the improvement of our study.
Round 3
Reviewer 4 Report
Dear authors, it seems to me that this manuscript has great relevance in the scientific world. I think the authors made the suggested changes, plus they considered the best writing options for a quality manuscript; therefore, I recommend approval.
