How the Inclusion of Pigeon Pea in Beef Cattle Diets Affects CH₄ Intensity: An In Vitro Fermentation Assessment

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper titled “How the inclusion of Pigeon pea in beef cattle diets affects CH4 intensity: an in vitro fermentation assessment” addresses an important and timely topic. The subject matter of the article is fascinating, and I read the manuscript with great interest. The paper aligns well with the scope of the journal and contributes valuable insights into the field of animal nutrition and greenhouse gas mitigation.

However, in its current form, the manuscript has several shortcomings that need to be addressed to improve the clarity, accuracy, and overall impact of the research. Specifically:

1.     Abstract and key words: I recommend rewriting the abstract and including more results and the significance of the obtained data. To enhance the research's appeal, I suggest avoiding the inclusion of terms in the keywords that are already present in the article title.

2.     Introduction: line 32. Consider citing 10.3389/fvets.2024.1441905. Moreover, I suggest making a greater introduction on in vitro techniques and gas production.

3.     Methodology Clarity: The description of the in vitro fermentation process is detailed, but it could benefit from additional clarification regarding the selection criteria for the inclusion levels of Pigeon pea in the diet. It would be helpful to explain why these specific levels (0%, 25%, 50%, 75%, and 100%) were chosen and how they relate to practical feeding strategies.

4.     Data Interpretation: While the results are presented clearly, there is a need for a more thorough discussion on the implications of the findings. For example, the observed increase in CH4 emission per gram of digestible dry matter (DMD) with higher Pigeon pea inclusion warrants a deeper exploration of the potential trade-offs between CH4 mitigation and DMD efficiency.

5.     Statistical Analysis: The manuscript mentions the use of statistical analysis but does not provide sufficient detail about the model selection process or the rationale behind choosing specific statistical tests. Including more information on this would strengthen the reliability of the conclusions drawn. The authors should consider including references to support the statistical methods used in the analysis. 

6.     Broader Implications: The paper would benefit from a discussion that situates the findings within the broader context of sustainable livestock management. How do these findings compare with other legume-based strategies for reducing enteric CH4 emissions? Are there practical recommendations for integrating Pigeon pea into existing cattle feeding programs?

7.     Literature Review: While the paper references relevant studies, the literature review could be expanded to include a more comprehensive overview of previous research on the effects of legumes on CH4 emissions and animal performance. This would help to better contextualize the study's contributions to the field.

8.     Conclusion: The conclusion provides a concise summary of the findings, but it could be enhanced by offering more specific recommendations for future research. For instance, what are the next steps in validating these results through in vivo studies, and how might these findings influence policy or practice in the field?

 

 

Author Response

Comments and Suggestions for Authors

The paper titled “How the inclusion of Pigeon pea in beef cattle diets affects CH4 intensity: an in vitro fermentation assessment” addresses an important and timely topic. The subject matter of the article is fascinating, and I read the manuscript with great interest. The paper aligns well with the scope of the journal and contributes valuable insights into the field of animal nutrition and greenhouse gas mitigation.

Thank you very much!

However, in its current form, the manuscript has several shortcomings that need to be addressed to improve the clarity, accuracy, and overall impact of the research. Specifically:

Comments 1: Abstract and key words: I recommend rewriting the abstract and including more results and the significance of the obtained data. To enhance the research's appeal, I suggest avoiding the inclusion of terms in the keywords that are already present in the article title.

Response 1: The suggestion was accepted. L13 – L29

 

Comments 2: Introduction: line 32. Consider citing 10.3389/fvets.2024.1441905. Moreover, I suggest making a greater introduction on in vitro techniques and gas production.

Response 2: The suggestion was accepted. L37 – L44

 

Comments 3: Methodology Clarity: The description of the in vitro fermentation process is detailed, but it could benefit from additional clarification regarding the selection criteria for the inclusion levels of Pigeon pea in the diet. It would be helpful to explain why these specific levels (0%, 25%, 50%, 75%, and 100%) were chosen and how they relate to practical feeding strategies.

Response 3: The suggestion was accepted. L96 – L102

 

Comments 4: Data Interpretation: While the results are presented clearly, there is a need for a more thorough discussion on the implications of the findings. For example, the observed increase in CH4 emission per gram of digestible dry matter (DMD) with higher Pigeon pea inclusion warrants a deeper exploration of the potential trade-offs between CH4 mitigation and DMD efficiency.

Response 4: The suggestion was accepted. L259 – L272

 

Comments 5: Statistical Analysis: The manuscript mentions the use of statistical analysis but does not provide sufficient detail about the model selection process or the rationale behind choosing specific statistical tests. Including more information on this would strengthen the reliability of the conclusions drawn. The authors should consider including references to support the statistical methods used in the analysis. 

Response 5: The suggestion was accepted. L152 – L158 and L169 – L176

 

Comments 6: Broader Implications: The paper would benefit from a discussion that situates the findings within the broader context of sustainable livestock management. How do these findings compare with other legume-based strategies for reducing enteric CH4 emissions? Are there practical recommendations for integrating Pigeon pea into existing cattle feeding programs?

Response 6: Yes, these recommendations exist, but it is a very new technology (little is known about this consortium). Embrapa, based on the commercialization of pigeon pea seeds, estimates that in the last 5 years 30 thousand hectares have been cultivated in consortium pasture systems, which is a small number when compared to the large amount of pastures in advanced stages of degradation in Brazil. With this in mind, we added the paragraph on the viability and applicability of the technology. L302 – L310

 

Comments 7: Literature Review: While the paper references relevant studies, the literature review could be expanded to include a more comprehensive overview of previous research on the effects of legumes on CH4 emissions and animal performance. This would help to better contextualize the study's contributions to the field.

Response 7: We tried to add this information, but we were unsuccessful in searching for new articles related to mixed pastures focused on mitigating CH₄ emissions. Main references: 34, 33, 25, 24, 10, 9, 8, 4, 3 and 2.

 

Comments 8: Conclusion: The conclusion provides a concise summary of the findings, but it could be enhanced by offering more specific recommendations for future research. For instance, what are the next steps in validating these results through in vivo studies, and how might these findings influence policy or practice in the field?

Response 8: Conclusion rewritten. L313 – L321

Reviewer 2 Report

Comments and Suggestions for Authors

This study aimed to evaluate the fermentation processes of tropical legume and grass, optimizing animal production and reducing greenhouse gas emissions. The result showed that the inclusion of 25% Pigeon pea in the diet was able to modulate in vitro fermentation, improving the fermentation and reducing energy loss. It is an interesting research work and will be useful for the readers of the journal. However, the manuscript also needs revisions. The detailed comments can be found in the attachment

 

Comments for author File: Comments.pdf

Author Response

Comments and Suggestions for Authors

This study aimed to evaluate the fermentation processes of tropical legume and grass, optimizing animal production and reducing greenhouse gas emissions. The result showed that the inclusion of 25% Pigeon pea in the diet was able to modulate in vitro fermentation, improving the fermentation and reducing energy loss. It is an interesting research work and will be useful for the readers of the journal. However, the manuscript also needs revisions. The detailed comments can be found in the attachment

Thank you very much!

 

This study aimed to evaluate the fermentation processes of tropical legume and grass, optimizing animal production and reducing greenhouse gas emissions. The result showed that the inclusion of 25% Pigeon pea in the diet was able to modulate in vitrofermentation, improving the fermentation and reducing energy loss. It is an interestingresearch work and will be useful for the readers of the journal. However, themanuscript also needs revisions. The detailed comments are as follows:

Abstract：

Comments 1: L19, The full names of DMD and DM should be added for the first time.

Response 1: The suggestion was accepted. L13 – L28

 

Comments 2: L19, reducing energy loss in the form of CH4, It's better to have it in digital form.

Response 2: The suggestion was accepted. L13 – L28

 

Comments 3: L22, TC, What does the abbreviation stand for.

Response 3: The suggestion was accepted. L13 – L28

 

Keywords:

Comments 4: L22, Brachiaria, Why is this in the keyword, but it doesn't appear anywhere else inthe

Response 4: The suggestion was accepted. L29

 

text. Introduction:

Comments 5: L45-49, Why are there references 7 and 9 on this side, followed by references 8.

Response 5: The suggestion was accepted, [8-10] = [8,9,10]. L57

 

Materials and Methods:

Comments 6: L85-86, bomb calorimeter, Please add the manufacturer and place of origin.

Response 6: The suggestion was accepted. L108

 

Comments 7: L89-91, What is the unit of condensed tannins.

Response 7: Eq. g leucocyanidin/kg of dry matter = g/kg of DM. L115

 

Comments 8: L102 and L115, pressure transducer and gas chromatography, Please add the placeof origin.

Response 8: The suggestion was accepted. L130 and L144 – L145

 

Results:

Comments 9: L149, it should be p<0.05

Response 9: The suggestion was accepted. L179

Discussion:

Comments 10: L201-203, Add references.

Response 10: The suggestion was accepted. L230 – L232

 

Comments 11: L232-233, as well as other legumes rich in condensed tannins (CT), When it first appears in the body, it has already written the full name and the short name, here onlywrite the short version.

Response 11: The suggestion was accepted. L260

 

Comments 12: L244-245, The following still does not explain clearly why the low NDF of leguminous forage in this study reduced the digestibility, please explain clearly.

Response 12: The suggestion was accepted. L270 – L281

 

References:

Comments 13: L305-307, animal, it should be Animal.

Response 13: The suggestion was accepted. L342 – L344

 

Comments 14: L360-362, The reference format should be unified, the full text is the abbreviations of journal titles, therefore, journal is referred to as AGROFORESTSYST.

Response 14: The suggestion was accepted. L358 – L360

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript analyzes the combination of tropical grasses with pigeon pea in different proportions in an in vitro study with the objective of understanding fermentation parameters and in particular methane production. 

The experiment is well planned with clear objectives and adequate methodology. However, it lacks originality, since it is a subject that has been studied relatively extensively. The fact that it is in combination with tropical grasses is perhaps worth mentioning.  Also, it is necessary to rewrite the conclusions, since it mentions other studies already published, and also includes a discussion of the conclusion, which is incorrect.   

Comments on the Quality of English Language

The quality of the writing is acceptable. 

Author Response

The manuscript analyzes the combination of tropical grasses with pigeon pea in different proportions in an in vitro study with the objective of understanding fermentation parameters and in particular methane production.

Thank you very much!

 

Comments 1: The experiment is well planned with clear objectives and adequate methodology. However, it lacks originality, since it is a subject that has been studied relatively extensively. The fact that it is in combination with tropical grasses is perhaps worth mentioning.  Also, it is necessary to rewrite the conclusions, since it mentions other studies already published, and also includes a discussion of the conclusion, which is incorrect.  

Response 1: The suggestion was accepted. L313 – L321

Reviewer 4 Report

Comments and Suggestions for Authors

This study investigated the effects of adding different proportions of pigeon pea to cattle feed on their ruminal fermentation process and methane emissions. The research team used in vitro fermentation technology to simulate the rumen environment of cattle and analyzed the effects of different proportions of pigeon pea addition on short-chain fatty acids, methane production, and dry matter digestibility.

 

General comment:

1.        The chemical composition data for pigeon peas must be provided, along with the nitrogen-free extract (NFE)% percentage and ash % in the diet containing pigeon peas.

2.        The study did not provide the protein digestibility data, but the CT effect on protein digestion showed considerable discussion in the manuscript.

 

General comment:

L22

The full name of “CT “ should be shown in the abstract

L61-65

The description of mixed culture in the trial should be clearer. Is the pigeon pea planted in the grassland that is already mixed with grass?

L75-76

What is the grazing simulation methodology? It should be a brief description of the operation.

L98-99

Was the pigeon pea applied in this study is removed from the pod hull before it put into the diet sample, or did the author apply a hole pod as the substrate?

L99-102

The author placed 1g of the substrate with 50 mL of inoculation liquid in a 250 mL bottle, leaving over 200 mL of empty space in each fermentation reaction. Was there any issue with gas pressure during the measurement process, such as it being too low or fluctuating unstably?

L110-111

Please explain why the author applied 144 h fermentation time but not 48 or 72 hr in this study.

After the process, the prolonged in vitro fermentation also displayed an atypical microbiota composition; the methane-producing bacteria exhibited relative weakness within the in vitro system.

L126-128

No “K” item was shown in the model at L125.

L131

The 5500 rpm is not the correct representation of centrifuge force. Please recalculate it as “x g”.

L151-153

Please explain the importance of K, Ti, and Yi values in the individual VFA production during fermentation.

L173 Table 4

Is there any NDF digestibility or CH4% of gas production data measured in this study? The author needs to explain the reason for the high GE but low digestibility of the high-level pigeon pea diets.

L182-185

The accumulated gas product of the 100% pigeon pea diet was much lower than that of other diets, but the decline of the CH4 production curve in Fig. 3A does not seem as much as the gas production in Fig. 3B. Why?

L185-187 and Fig.4

How about the coefficient of determination (R²) and its p-value in Fig.4 ? why did the CH4 production decrease with the increase of CP% in diet?

L237

…consequently, animal performance [25, 26].

Which performance was affected?

L240-242

Is there any second metabolic compound that was assayed in this study?

L245-247

The cell wall composition was different between grass and legume. The legume also had higher ADF%, but the NDF digestibility of the legume sometimes was better than that of grass. Without the NDF digestibility data, the discussion in this section should be reconsidered.

L258-261

According to the description in this section, does it mean that the in vitro test was unsuitable for evaluating the high TC-containing diet like the diets in this study?

L282-284

The present data seem insufficient to support the amino acid absorption after feeding in this study.

Author Response

Comments and Suggestions for Authors

This study investigated the effects of adding different proportions of pigeon pea to cattle feed on their ruminal fermentation process and methane emissions. The research team used in vitro fermentation technology to simulate the rumen environment of cattle and analyzed the effects of different proportions of pigeon pea addition on short-chain fatty acids, methane production, and dry matter digestibility.

 

General comment:

Comments 1: The chemical composition data for pigeon peas must be provided, along with the nitrogen-free extract (NFE)% percentage and ash % in the diet containing pigeon peas.

Response 1: The most common food analysis system, the Weende system or proximal system, does not have a specific determination of non-structural carbohydrates, but it has an approximation which is the nitrogen-free extract (NFE). In fact, the NFE is the total DM subtracted from the sum of the determined values ​​of Crude Protein, Ether Extract, Crude Fiber and Ash. The NFE includes all the errors of these analyses. The largest of these would be in the crude fiber fraction which results in an overestimation of the NFE. Crude fiber is being replaced in practically all animal nutrition laboratories by Van Soest's Neutral Detergent Fiber (NDF). Thereby, the crude fiber is theoretically formed by cellulose, alkali insoluble lignin, and residues of hemicellulose. So, much of the hemicellulose and lignin is included in NFE. This fraction (which was supposed to contain the easily digested carbohydrates) presents low and variable digestibility and together with underestimation of fibrous compounds constitute the main functional limitation for using crude fiber and NFE in ruminant nutrition. L110 – L112 and L114 – L117

 

Comments 2: The study did not provide the protein digestibility data, but the CT effect on protein digestion showed considerable discussion in the manuscript.

Response 2: The suggestion was accepted. L282 – L289 and L313 – L321

 

General comment:

Comments 3: L22

 The full name of “CT “ should be shown in the abstract

Response 3: The suggestion was accepted. L13 – L28

 

Comments 4: L61-65

The description of mixed culture in the trial should be clearer. Is the pigeon pea planted in the grassland that is already mixed with grass?

Response 4: Yes, the pasture was initially cultivated with Urochloa decumbens cv. Basilisk and Urochloa brizantha cv. Marandu. The legume was later planted in rows in the area. Sampling was carried out individually. L73 – L77

 

Comments 5: L75-76

What is the grazing simulation methodology? It should be a brief description of the operation.

Response 5: The suggestion was accepted. L88 – L94

 

Comments 6: L98-99

Was the pigeon pea applied in this study is removed from the pod hull before it put into the diet sample, or did the author apply a hole pod as the substrate?

Response 6: The suggestion was accepted. L91 – L92

 

Comments 7: L99-102

The author placed 1g of the substrate with 50 mL of inoculation liquid in a 250 mL bottle, leaving over 200 mL of empty space in each fermentation reaction. Was there any issue with gas pressure during the measurement process, such as it being too low or fluctuating unstably?

Response 7: The suggestion was accepted. No, all fermenters produced gas at all times, except for the white flasks (they did not contain 1 gram of sample), at 12 hours the fermentation produced only 5 ml of gas and it went to zero at 24 hours of fermentation. This was expected in this situation. All other fermenters finished 144 hours producing a gas volume of approximately 16 ml.

 

Comments 8: L110-111

Please explain why the author applied 144 h fermentation time but not 48 or 72 hr in this study.

After the process, the prolonged in vitro fermentation also displayed an atypical microbiota composition; the methane-producing bacteria exhibited relative weakness within the in vitro system.

Response 8: It was necessary for the curve to be inflected to generate reliable results, and since these are tropical plants (rich in fiber), we decided to do it up to 144 h.

 

Comments 9: L126-128

No “K” item was shown in the model at L125.

Response 9: In ruminant nutrition the variable C is called K representing the rate.

L152 – L158

 

Comments 10: L131

The 5500 rpm is not the correct representation of centrifuge force. Please recalculate it as “x g”.

Response 10: The suggestion was accepted. L161

 

Comments 11: L151-153

Please explain the importance of K, Ti, and Yi values in the individual VFA production during fermentation.

Response 11: The suggestion was accepted. L152 – L158 and L181 – L185

 

Comments 12: L173 Table 4

Is there any NDF digestibility or CH4% of gas production data measured in this study? The author needs to explain the reason for the high GE but low digestibility of the high-level pigeon pea diets.

Response 12: Being justified by the presence of secondary compounds interfering in the in vitro fermentation process. L276 – L279

 

Comments 13: L182-185

The accumulated gas product of the 100% pigeon pea diet was much lower than that of other diets, but the decline of the CH4 production curve in Fig. 3A does not seem as much as the gas production in Fig. 3B. Why?

Response 13: The suggestion was accepted. L200 – L202; L211 – L213 and L204 – L208

 

Comments 14: L185-187 and Fig.4

How about the coefficient of determination (R²) and its p-value in Fig.4 ? why did the CH4 production decrease with the increase of CP% in diet?

Response 14: The suggestion was accepted. L233 – L237

 

Comments 15: L237

…consequently, animal performance [25, 26].

Which performance was affected?

Response 15: The suggestion was accepted. L264

 

Comments 16: L240-242

Is there any second metabolic compound that was assayed in this study?

Response 16: It was not the focus of the article, however it is a good observation for future work. Thank you very much.

 

Comments 17: L245-247

The cell wall composition was different between grass and legume. The legume also had higher ADF%, but the NDF digestibility of the legume sometimes was better than that of grass. Without the NDF digestibility data, the discussion in this section should be reconsidered.

Response 17: The suggestion was accepted. L270 – L281 and L313 – L321

 

Comments 18: L258-261

According to the description in this section, does it mean that the in vitro test was unsuitable for evaluating the high TC-containing diet like the diets in this study?

Response 18: The suggestion was accepted. L288 – L289

 

Comments 19: L282-284

The present data seem insufficient to support the amino acid absorption after feeding in this study.

Response 19: OK, we withdraw that observation. L313 – L321

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

the paper improved a lot, I do not have any further change

Author Response

The paper improved a lot, I do not have any further change.

 

We appreciate your immense contribution to this work..Thank you very much!

Reviewer 4 Report

Comments and Suggestions for Authors

L104

The AOAC Method 923.03, is the assay method for Ash, not for mineral material. Please correct the item in the text and Table 1.

 

L155  "B, the mathematical constant of the mode."

Please confirm that label B in Figure 2 is correct. The present B point seems to mean the "lag time" in the model.

Author Response

L104

The AOAC Method 923.03, is the assay method for Ash, not for mineral material. Please correct the item in the text and Table 1.

The change was made. L104, L105 and L106

 

L155  "B, the mathematical constant of the mode."

Please confirm that label B in Figure 2 is correct. The present B point seems to mean the "lag time" in the model.

The change was made. L.155 and L156

 

 

We appreciate your immense contribution to this work. Thank you very much!