Effect of Dietary Calcium Nitrate Addition on Methane Emission, Nitrogen Excretion, and Ruminal Fermentation Parameters and Microbiota in Liuyang Black Goats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor and Authors,

This manuscript examines the impact of dietary calcium nitrate supplementation (3% DM basis) on the composition of the rumen microbiota, nitrogen metabolism, methane and CO₂ emissions, and ruminal fermentation parameters in Liuyang black goats. This research covers a significant issue, which is the mitigation of enteric methane from ruminants. This topic is particularly relevant to the sustainable production of livestock as well as techniques for mitigating the effects of climate change. The study provides a multi-level approach to studying the effects of calcium nitrate by combining measures of gas emission with investigation of fermentation in the rumen and sequencing of the 16S rRNA gene. Respiration calorimetry is a valid and trustworthy method for measuring methane. The topic is timely, given the global emphasis on lowering livestock greenhouse gas emissions. However, the article has some important flaws that must be resolved before it can be considered for publication.

Best regrds.

Major comments:

L40-41, L303-320: The abstract states: ‘...lower ammonia nitrogen (NH₃-N) concentration 3 hours post-feeding (p < 0.05).’ However, the results table for rumen fermentation parameters shows that NH₃-N at 3h post-feeding was actually HIGHER in the CAL group (15.434 mg/100 mL) compared to CON (13.461 mg/100 mL), with p = 0.184 (NOT significant). This is a direct contradiction that undermines the credibility of the manuscript.

L74-79: The introduction appropriately discusses nitrate toxicity concerns and methemoglobin formation, yet no blood parameters or methemoglobin levels were monitored during the experiment. Given that 3% calcium nitrate supplementation is a relatively high dose, monitoring blood methemoglobin is essential for evaluating animal safety and for the responsible recommendation of this feeding strategy.

If blood samples were collected, include methemoglobin data. If not, this must be acknowledged as a major limitation and the conclusion about ‘promoting environmental sustainability’ should be tempered accordingly. At minimum, clinical observations of animals during the trial (signs of toxicity, feed refusal, etc.) should be reported.

L103-113: The experimental design description is confusing and raises pseudoreplication concerns. The authors state that 12 goats were divided into two groups (n=6), but the experiment was conducted in two phases with only 3 goats per group per phase. It is unclear whether the same goats were used in both phases or if different animals were assigned.

L118-124: The two diets are not balanced for nitrogen source. The CAL diet replaces expanded urea (0.80%) and calcium carbonate (0.25%) with calcium nitrate (3.00%), while also changing soybean meal content substantially (11.35% vs. 7.55%) and corn content (48.60% vs. 49.65%). This creates multiple confounding variables. The CP levels (14.34% vs. 14.70%) are relatively close but the nitrogen source profile is fundamentally different (urea vs. nitrate, higher vs. lower SBM). Additionally, the calcium content differs substantially (0.31% vs. 0.81% Ca), which could independently affect rumen fermentation.

Therefore, please discuss these confounding variables as a limitation. Consider whether the Ca difference could affect rumen pH and fermentation independently. Justify the diet formulation strategy and explain why isonitrogenous diets with equal urea/NPN content were not used as controls.

L125, L274, L290, L300, L317, L354, L357: All data tables throughout the manuscript are labeled as ‘Table 1’. Each should have a unique sequential number.

L223-244: Two different statistical approaches are described but neither adequately handles the experimental structure: (a) The linear model (Yᵢ = μ + Tᵢ + βₜ·tⱼ + εᵢ) treats the 5-day collection period means as single observations per group but includes a time covariate that is inappropriate for only two time stages with minimal replication within each. (b) The independent t-test is used for most parameters, but this ignores the phase structure entirely. Furthermore, rumen fermentation parameters were measured at two time points (before feeding and 3h post-feeding), representing repeated measures on the same animals, yet these are analyzed independently. No mention is made of checking normality assumptions, homogeneity of variance, or how outliers were handled.

L272-293: The CH₄/ADFI value is notably HIGHER in the CAL group (43.021 g/kg ADFI) compared to CON (36.316 g/kg ADFI) with p = 0.396. This means that per unit of acid detergent fiber intake, the calcium nitrate group actually produced more methane. This contradicts the overall narrative that calcium nitrate reduces methane. Please discuss all CH₄ emission metrics transparently, including those that are non-significant or contradict the main finding. Explain why CH₄/ADFI showed the opposite trend.

L331, L336: Similarly, figures are inconsistently numbered—’Figure 1’ is used for both the alpha diversity boxplots and the PCoA plot.

L416-418: ‘lower CH₄ emissions were associated with changes in DMI and ADG, which differed significantly between treatment groups.’ However, Table shows DMI and ADG were NOT significantly different (p > 0.05). This is contradictory.

L538-546: The conclusion is overly optimistic given that nitrogen metabolism was not affected, growth performance was not improved, and several CH₄ metrics were not significant. The statement about ‘enhancing feed efficiency’ is not supported by the data.

Minor comments:

Keywords should include ‘greenhouse gas’ and ‘16S rRNA’ to improve discoverability.

L24-25: ‘the pH level in their stomachs’ should be ‘rumen pH’.

L80: ‘methemoglobins formation’ should be ‘methemoglobin formation’

L100: ‘goats production’ should be ‘goat production.’

L207-208: QIIME version 1.9.1 is outdated (released 2015). Consider whether QIIME2 would have been more appropriate, or justify the use of version 1.

L223: ‘Statistical Analys’ should be ‘Statistical Analysis.’

Author Response

Dear reviewer,

We are very grateful to Reviewer for reviewing the paper so carefully.

We have tried our best to improve the manuscript and have modified some. Specific changes are as follows:

Re: Manuscript ID: animals-4184893 and Title: Effect of dietary calcium nitrate addition on methane emission, nitrogen excretion, and ruminal fermentation parameters and microbiota in Liuyang black goats.

Comments and Suggestions for Authors

Revision 1. The abstract states: ‘...lower ammonia nitrogen (NH₃-N) concentration 3 hours post-feeding (p < 0.05).’ However, the results table for rumen fermentation parameters shows that NH₃-N at 3h post-feeding was actually HIGHER in the CAL group (15.434 mg/100 mL) compared to CON (13.461 mg/100 mL), with p = 0.184 (NOT significant). This is a direct contradiction that undermines the credibility of the manuscript.

Response 1: We sincerely thank the reviewers for identifying this fundamental and critical issue and apologize for any inconvenience caused during the review process. In the original abstract, the NH₃-N concentration was incorrectly reported as lower than that of the control group; in fact, it was higher. The abstract has since been deleted and revised accordingly. We apologize for the oversight in this aspect in the original text and have conducted a systematic review and supplementation based on your suggestions. (Lines 40-41，Lines303-320).

Revision 2. The introduction appropriately discusses nitrate toxicity concerns and methemoglobin formation, yet no blood parameters or methemoglobin levels were monitored during the experiment. Given that 3% calcium nitrate supplementation is a relatively high dose, monitoring blood methemoglobin is essential for evaluating animal safety and for the responsible recommendation of this feeding strategy.

Response 2: Thank you for this pertinent comment. We fully agree that, at the 3 % calcium-nitrate inclusion used here, serial measurement of blood methemoglobin (MetHb) is indispensable for assessing animal safety. Because the original protocol focused on growth performance, feed intake and rumen microbiota, MetHb was not monitored—an acknowledged limitation. Nevertheless, our dosing schedule was built on the consensus that gradual adaptation minimizes the risk of nitrate/nitrite toxicity. As previously demonstrated, Halmemies et al. fed 5.0% calcium nitrate (≈21 g NO₃⁻/kg DM) to 10 kg goats for 12 weeks without reducing daily weight gain or daily feed intake (Ref. 9); Van Zijderveld et al. reported no adverse effects in 43 kg lambs supplemented with 26 g CaNO₃/kg DM (≈17 g NO₃⁻/kg DM) for 4 weeks (Ref. 37); Seyyedsalehi et al. established a safety threshold for sheep of <30 g NO₃⁻/kg DM (Ref. 38). The nitrate content in the experimental diet was 19.4 g/kg DM (Table 2), 35% below this toxicity threshold. Li et al. observed a 19% reduction in daily weight gain compared to the urea group at a nitrate level of 21 g/kg DM, though no change in feed intake was noted (Ref. 10). Based on these studies, this dosage was selected for the relevant trials.(Lines 74-79).

Revision 3. The experimental design description is confusing and raises pseudoreplication concerns. The authors state that 12 goats were divided into two groups (n=6), but the experiment was conducted in two phases with only 3 goats per group per phase. It is unclear whether the same goats were used in both phases or if different animals were assigned.

Response 3: We sincerely thank the reviewers for raising this fundamental and crucial point. Two treatments were arranged, each with six goats, and the individual animal served as the experimental unit. Owing to pen limitations, the trial was executed in two consecutive batches, with three goats per treatment per batch. All six goats within a treatment were maintained under identical husbandry conditions throughout: the first three were sampled in period 1 and the remaining three in period 2; this detail has now been incorporated into the revised manuscript. (Lines 103-113)

Revision 4. The two diets are not balanced for nitrogen source. The CAL diet replaces expanded urea (0.80%) and calcium carbonate (0.25%) with calcium nitrate (3.00%), while also changing soybean meal content substantially (11.35% vs. 7.55%) and corn content (48.60% vs. 49.65%). This creates multiple confounding variables. The CP levels (14.34% vs. 14.70%) are relatively close but the nitrogen source profile is fundamentally different (urea vs. nitrate, higher vs. lower SBM). Additionally, the calcium content differs substantially (0.31% vs. 0.81% Ca), which could independently affect rumen fermentation.

Response 4: Thank you for pointing out that differences in nitrogen sources, calcium concentration, and raw material composition between CAL and CON feeds may introduce confounding factors. We fully acknowledge these are not “single-factor” variables. We will pay particular attention to such confounding factors in future trial designs. The use of calcium nitrate in this trial reflects practical farm conditions, where producers typically do not purchase 46% crystalline urea to precisely adjust nitrogen levels nor deliberately reduce soybean meal usage to offset additional calcium loading. Strict adherence to equal nitrogen and calcium principles would be impractical under real-world cost constraints and would increase feed expenses. Furthermore, previous literature (Li et al., ref. 10) indicates that animals receiving 3% calcium nitrate exhibit similar performance to those fed 1.5% urea as a fermentable nitrogen source. (lines 118-124)

Revision 5. L125, L274, L290, L300, L317, L354, L357: All data tables throughout the manuscript are labeled as ‘Table 1’. Each should have a unique sequential number.

Response 5: Thank you for pointing this out. We have now re-numbered all data tables throughout the manuscript sequentially as Table 1, Table 2, Table 3, etc., and cross-checked every in-text citation, figure legend, and appendix reference to ensure complete consistency. We sincerely thank the reviewers for identifying this fundamental and critical issue and apologize for any inconvenience caused during the review process.  (Line125, Line274, Line290, Line300, Line317, Line354, Line357)

Revision 6. L223-244: Two different statistical approaches are described but neither adequately handles the experimental structure: (a) The linear model (Yᵢ = μ + Tᵢ + βₜ·tⱼ + εᵢ) treats the 5-day collection period means as single observations per group but includes a time covariate that is inappropriate for only two time stages with minimal replication within each. (b) The independent t-test is used for most parameters, but this ignores the phase structure entirely. Furthermore, rumen fermentation parameters were measured at two time points (before feeding and 3h post-feeding), representing repeated measures on the same animals, yet these are analyzed independently. No mention is made of checking normality assumptions, homogeneity of variance, or how outliers were handled.

Response 6: We thank the reviewer for the thorough statistical comments and for highlighting potential limitations in our analytical approach. We acknowledge that treating the 5-day collection-period means as single observations per group is a simplification. Because replication within each stage was limited, we included a time covariate to account for possible temporal trends. We recognize that with only two time points a linear model may not fully capture stage-specific variation; this limitation has now been clarified in the revised Methods section.For most parameters we used independent-samples t-tests to provide straightforward between-group comparisons. We agree that this approach does not explicitly account for the stage or repeated-measures structure. For rumen-fermentation parameters, measured pre-feeding and 3 h post-feeding, we now acknowledge that these are repeated measures. Although the small sample size precluded more complex mixed-model analyses, we have added a statement indicating that assumptions of normality, homogeneity of variances, and the presence of outliers were examined (Shapiro–Wilk test, Levene’s test, and visual inspection of residuals, respectively).We appreciate the reviewer’s suggestions and have revised the manuscript to explicitly acknowledge these statistical limitations and their potential impact on interpretation. (Lines 223-244)

Revision 7. L272-297: The CH₄/ADFI value is notably HIGHER in the CAL group (43.021 g/kg ADFI) compared to CON (36.316 g/kg ADFI) with p = 0.396. This means that per unit of acid detergent fiber intake, the calcium nitrate group actually produced more methane. This contradicts the overall narrative that calcium nitrate reduces methane. Please discuss all CH₄ emission metrics transparently, including those that are non-significant or contradict the main finding. Explain why CH₄/ADFI showed the opposite trend.

Response 7: We sincerely thank the reviewers for identifying this fundamental and critical issue and apologize for any inconvenience caused during the review process. In the original abstract, the CH₄/ADFI value was incorrectly stated as significantly higher in the CAL group (43.021 g/kg) than in the CON group (36.316 g/kg); in fact, it was the CON group (49.461 g/kg) that exhibited the higher value compared with the CAL group (39.828 g/kg). This has been corrected in both the abstract and main text. We deeply regret this oversight and have conducted a comprehensive review and revision based on your suggestions. (Lines 272-297).

Revision 8. L331, L336: Similarly, figures are inconsistently numbered—’Figure 1’ is used for both the alpha diversity boxplots and the PCoA plot.

Response 8: Thank you for pointing this out. We have now corrected the inconsistency: the PCoA plot that was previously labeled “Figure 1” has been re-numbered “Figure 2”, and all subsequent figures and in-text citations have been updated accordingly. (Lines L331, L336)

Revision 9. L422-424: ‘lower CH₄ emissions were associated with changes in DMI and ADG, which differed significantly between treatment groups.’ However, Table shows DMI and ADG were NOT significantly different (p > 0.05). This is contradictory.

Response 9: Thank you for pointing out this discrepancy. Upon verification, we found inaccuracies in the original text on lines 416–418. The actual situation is as follows: although CH₄ emissions decreased significantly across all groups, the p-values for both DMI and ADG were >0.05, failing to reach statistical significance. The original text has been revised accordingly. We apologize for this oversight and have implemented systematic revisions and additions based on your suggestions. (Lines 422-424)

Revision 10. L538-546: The conclusion is overly optimistic given that nitrogen metabolism was not affected, growth performance was not improved, and several CH₄ metrics were not significant. The statement about ‘enhancing feed efficiency’ is not supported by the data.

Response 10: Thank you for this valuable comment. We have carefully re-examined the original statements on feed efficiency in the Conclusion and have rewritten and removed them accordingly. The revised text now strictly reflects the statistically non-significant results reported in the manuscript, thereby avoiding any over-interpretation. We sincerely appreciate the reviewer’s rigorous scrutiny, which has helped us improve the objectivity and accuracy of our conclusions. (Lines 538-546)

Revision 11. Keywords should include ‘greenhouse gas’ and ‘16S rRNA’ to improve discoverability.
Response 11: Thank you for this helpful suggestion. We have added both “greenhouse gas” and “16S rRNA” to the Keywords list to enhance the manuscript’s discoverability.

Revision 12. L24-25: ‘the pH level in their stomachs’ should be ‘rumen pH.

Response 12: Thank you for this helpful suggestion. We have revised lines 24–25, replacing “the pH level in their stomachs” with “rumen pH” to employ the precise anatomical terminology. (Lines 24-25)

Revision 13. L73: ‘methemoglobins formation’ should be ‘methemoglobin formation’.

Response 13: Thank you for pointing out this typo. We have corrected “methemoglobins formation” to “methemoglobin formation” on line 80. (Line 73)

Revision 14. L101: ‘goats production’ should be ‘goat production.

Response 14: Thank you for catching this grammatical error. We have corrected “goats production” to “goat production” on Line100. (Lines 101)

Revision 15. L207-208: QIIME version 1.9.1 is outdated (released 2015). Consider whether QIIME2 would have been more appropriate, or justify the use of version 1.

Response 15: Thank you for this observation. We fully agree that QIIME2 represents a major advance over QIIME 1.9.1. Nevertheless, our 16S rRNA pipeline was established during an earlier project and the raw reads had already been processed with QIIME 1.9.1. To maintain comparability with our previously published data, we retained this upstream version. We appreciate the reviewers' suggestions for enhancing the professionalism and rigor of the manuscript..(Lines 207-208)

Revision 16.L223: ‘Statistical Analys’ should be ‘Statistical Analysis.

Response 16: Thank you for catching this typo. We have corrected the heading to “Statistical Analysis” in the revised manuscript. Special thanks to the reviewer for their meticulous review and valuable suggestions, which have significantly enhanced the academic rigor and quality of the original manuscript. (Line 223).

Reviewer 2 Report

Comments and Suggestions for Authors

1、Is there any reference basis for the authors to add 3% calcium nitrate in the goat diet? Why was only one supplemental concentration set? I believe the authors should provide an explanation.

2、The authors should calculate the metabolizable energy (ME) of the diet.

3、Compared with the control group, the ADG of the CAL group was increased by 12%, but the difference was not statistically significant. The authors should provide a reasonable explanation; could this be due to large individual variation among goats within the group?

4、I note that the authors weighed the goats daily when measuring growth performance. I question whether this is appropriate, as I believe it would impose continuous stress on the goats.

5、I note that dry matter intake, nutrient digestibility, and total volatile fatty acid production were all decreased following the addition of 3% calcium nitrate. It is necessary to consider whether the 3% inclusion level was excessively high and adversely affected animal performance.

Author Response

Dear reviewer,

We are very grateful to Reviewer for reviewing the paper so carefully.

We have tried our best to improve the manuscript and have modified some. Specific changes are as follows:

Re: Manuscript ID: animals-4184893 and Title: Effect of dietary calcium nitrate addition on methane emission, nitrogen excretion, and ruminal fermentation parameters and microbiota in Liuyang black goats.

Comments and Suggestions for Authors

Revision 1. Is there any reference basis for the authors to add 3% calcium nitrate in the goat diet? Why was only one supplemental concentration set? I believe the authors should provide an explanation..

Response 1: We appreciate this insightful comment. The 3 % (w/w, as-fed) calcium-nitrate level was chosen after scaling the “safe and effective” nitrate-N doses reported in peer-reviewed goat and lamb studies to a 28 kg goat grower pellet. As previously demonstrated, Halmemies et al. fed 5.0% calcium nitrate (≈21 g NO₃⁻/kg DM) to 10 kg goats for 12 weeks without reducing daily weight gain or daily feed intake (Ref. 9); Van Zijderveld et al. reported no adverse effects in 43 kg lambs supplemented with 26 g CaNO₃/kg DM (≈17 g NO₃⁻/kg DM) for 4 weeks (Ref. 37); Seyyedsalehi et al. established a safety threshold for sheep of <30 g NO₃⁻/kg DM (Ref. 38). The nitrate content in the experimental diet was 19.4 g/kg DM (Table 2), 35% below this toxicity threshold. Li et al. observed a 19% reduction in daily weight gain compared to the urea group at a nitrate level of 21 g/kg DM, though no change in feed intake was noted (Ref. 10). Based on these studies, this dosage was selected for the relevant trials.

Revision 2. The authors should calculate the metabolizable energy (ME) of the diet.

Response 2: We thank the reviewer for recommending the determination of metabolizable energy (ME); we fully concur with this suggestion. Regrettably, our animal facility is not equipped with open-circuit respiration chambers nor the trained personnel required for total-collection digestibility trials, so we were unable to obtain the in-vivo data necessary to calculate true ME. Should the requisite infrastructure become available in the future, we will conduct a formal digestion/energy-balance study to acquire accurate ME values. We apologize for this oversight in the original manuscript and have now systematically reviewed and incorporated your suggestions into the design of forthcoming experiments.

Revision 3. Compared with the control group, the ADG of the CAL group was increased by 12%, but the difference was not statistically significant. The authors should provide a reasonable explanation; could this be due to large individual variation among goats within the group?

Response 3: We appreciate the reviewer's suggestion and fully agree with your perspective. The 12% increase in daily weight gain in the CAL group did not reach statistical significance between groups. This limitation primarily stems from the experimental design: the open-air respiration chamber could only accommodate a limited number of animals at a time, resulting in a small sample size. Therefore, the lack of significance likely reflects insufficient statistical power due to excessive inter-individual variation rather than an absence of biological effect. In future studies, we will rigorously control for initial weight homogeneity and expand sample sizes to reduce variability. We appreciate the reviewer's suggestion and will enhance the manuscript's rigor and scientific merit accordingly.

Revision 4. I note that the authors weighed the goats daily when measuring growth performance. I question whether this is appropriate, as I believe it would impose continuous stress on the goats.

Response 4: We sincerely appreciate your valuable suggestions and fully acknowledge your concerns. This study has implemented measures such as a 10-day adaptation period, group-synchronized weighing, and dedicated training personnel to minimize stress responses. Relevant details have now been incorporated into the revised manuscript. Following your recommendation, all subsequent trials have been adjusted to triennial weighing intervals and incorporate unattended automated scales, completely eliminating the possibility of cumulative stress. Your reminder has prompted us to re-examine and reinforce our commitment to balancing scientific rigor with animal welfare, for which we are deeply grateful.

Revision 5. I note that dry matter intake, nutrient digestibility, and total volatile fatty acid production were all decreased following the addition of 3% calcium nitrate. It is necessary to consider whether the 3% inclusion level was excessively high and adversely affected animal performance.

Response 5: We sincerely appreciate your valuable suggestion and the reminder to reconsider whether the 3 % inclusion level was excessive. We fully share your concern. In the revised manuscript we have added a prospective statement describing a follow-up dose–response study that will evaluate 0 %, 1 %, 2 % and 3 % calcium nitrate in a step-wise manner, while continuously monitoring dry-matter intake, rumination time and blood MetHb to safeguard both animal performance and welfare. Thank you once again for your meticulous review and constructive advice, which have prompted us to explore the dose effect in greater depth and to refine our future experimental design. Special thanks to the reviewer for their meticulous review and valuable suggestions, which have significantly enhanced the academic rigor and quality of the original manuscript.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

This study aimed to evaluate the effect of adding calcium nitrate to goats' diets. The manuscript's concept is worth exploring and could yield important findings in animal nutrition. However, some concerns need to be addressed.

Line 28: According to the results section, nutrient digestibility did not improve; please rewrite according to the results

Line 35: Please run a power test analysis to validate whether 6 animals per treatment is enough to draw a good conclusion

Line 36: Which phases? Please explain them in the abstract

Lines 36-37: There is a duplication here, please delete

Lines 37-39: repeated information in the same statement, please rewrite

Lines 47-78: None of the results mentioned in the abstract showed an improvement in feed efficiency. Additionally, the results section indicates that feed efficiency did not differ between the two treatments.

Line 49: The abstract lacks some data. Please add more data

Lines 54-80: This section is well-written, but I believe it is too long. Please shorten it

Line 94: Before this line, please insert more literature about the use of calcium nitrate in the diets of livestock

Lines 96-100: please revise the hypothesis for better clarity and accuracy.

Line 108: Please add this information to the abstract

Lines 103-107: In total, using 12 animals (6 per treatment) and using two phases with 3 animals per group is low, causing statistical power is limited

Line 115: housed individually!! Please clarify

Line 116: ad libitum!!! Please clarify

Line 126: In Table 1, the calcium level is higher in the treatment group. Don’t you think this could influence the results?

Lines 137-138: Is there a specific position in the pens allowed to collect all fecal output?

Lines 147-148: How did you collect the rumen fluid? Tube!!!

Lines 155-156: Could you please explain the calibration procedure and recovery rate validation? Did you adapt the animals to the chambers?

Line 172: replace “dry matter” with “DM”

Line 172: replace “crude protein” with “CP”

Lines 223-244: This section is not really clear to the readers. Please rewrite

Lines 262 and 274: Rename this Table to be “Table 2.” Also, other tables need to be labeled correctly

Lines 268-269: Results of ADG and DMI were similar, so how do you explain the large CO2 reduction since it's linked to metabolic rate? 

Line 290: In this Table, SEM is too low for ADG. Please recheck

Lines 295-299: nitrogen balance data did not differ between the treatments. However, the abstract and introduction imply improved N utilization. This is not consistent.

Line 311: The results indicate that Nh3-N is decreased, but in the discussion (lines 480-484), the authors state it is increased. Please clarify this contradiction.

Line 541: This statement is not correct: “may enhance….utilization” Please fix it

 

 

 

Author Response

Dear reviewer,

We are very grateful to Reviewer for reviewing the paper so carefully.

We have tried our best to improve the manuscript and have modified some. Specific changes are as follows:

Re: Manuscript ID: animals-4184893 and Title: Effect of dietary calcium nitrate addition on methane emission, nitrogen excretion, and ruminal fermentation parameters and microbiota in Liuyang black goats.

Comments and Suggestions for Authors

Revision 1. Line 28: According to the results section, nutrient digestibility did not improve; please rewrite according to the results.

Response 1: We sincerely thank the reviewers for pointing out this fundamental critical issue and deeply apologize for any inconvenience caused during the review process. In the original abstract, the digestibility of nutrients was not improved. The abstract has now been deleted and revised accordingly. We apologize for the omission in the original text and have systematically revised and supplemented it based on your suggestions (Lines 28).

Revision 2. Please run a power test analysis to validate whether 6 animals per treatment is enough to draw a good conclusion.

Response 2: We sincerely appreciate the reviewer's suggestions and fully agree with your perspective. Conducting a power analysis confirms that six sheep can yield reliable conclusions, which we have incorporated into the manuscript. Additionally, we note that similar results can be obtained by following the methodology previously described by Wang et al. (Reference 10). We extend our gratitude once again for the reviewer's valuable input, which has significantly enhanced the quality of this manuscript.( Line35)

Revision 3. Which phases? Please explain them in the abstract.

Response 3: We are particularly grateful for the reviewer's suggestions. We fully agree with your perspective and have already incorporated the revisions into the original abstract. Once again, we extend our sincere thanks to the reviewer for enhancing the quality of this manuscript.(Lines36)

Revision 4. There is a duplication here, please delete.

Response 4: We sincerely appreciate the reviewer's suggestions and apologize for the oversight. We have verified the abstract and made the necessary revisions in the original abstract. Once again, we thank the reviewer for enhancing the quality of the manuscript. (Lines36-37)

Revision 5. repeated information in the same statement, please rewrite.

Response 5: We have incorporated the reviewers' suggestions into the revised manuscript. We appreciate the reviewers' recommendations for enhancing the quality and academic rigor of the manuscript. (Lines37-39)

Revision 6. None of the results mentioned in the abstract showed an improvement in feed efficiency. Additionally, the results section indicates that feed efficiency did not differ between the two treatments.

Response 6: Thank you for this valuable comment. We have carefully re-examined the original statements on feed efficiency in the Conclusion and have rewritten and removed them accordingly. The revised text now strictly reflects the statistically non-significant results reported in the manuscript, thereby avoiding any over-interpretation. We sincerely appreciate the reviewer’s rigorous scrutiny, which has helped us improve the objectivity and accuracy of our conclusions. (Lines 47-48).

Revision 7. The abstract lacks some data. Please add more data.

Response 7: Thank you for your valuable feedback. We have revised the abstract section accordingly. We sincerely appreciate the reviewer's thorough review, which has helped us enhance the logical coherence of the manuscript. (Line 49)

Revision 8. This section is well-written, but I believe it is too long. Please shorten it.

Response 8: We are particularly grateful for the reviewer's suggestions. We fully agree with your perspective and have incorporated the revisions into the original paper's introduction. Once again, we sincerely thank the reviewer for enhancing the quality of this manuscript. (Lines54-80)

Revision 9. Before this line, please insert more literature about the use of calcium nitrate in the diets of livestock.

Response 9: We sincerely appreciate the reviewer's suggestions and have incorporated the relevant content. Once again, we thank the reviewer for enhancing the quality of the manuscript. (Lines 94)

Revision 10. please revise the hypothesis for better clarity and accuracy.

Response 10: We sincerely appreciate the reviewer's suggestions and fully agree with them. We have revised the hypotheses accordingly. Once again, we thank the reviewer for their contributions to enhancing the quality of this manuscript. (Lines96-100)

Revision 11. Please add this information to the abstract.

Response 11: We sincerely appreciate the reviewer's suggestions and fully agree with these comments. We have incorporated them into the abstract. Once again, we thank the reviewer for their contributions to enhancing the quality of this manuscript. (Lines 108).

Revision 12. In total, using 12 animals (6 per treatment) and using two phases with 3 animals per group is low, causing statistical power is limited.

Response 12: We sincerely appreciate the reviewer for raising this fundamental and critical question. We fully agree with the reviewer's suggestion to collect samples from only six sheep. This limitation stems from the high cost of constructing the facilities required for respiratory calorimetry. In future experiments, we will expand the sample size and increase the number of sheep collected. We again thank the reviewer for their suggestions, which have significantly enhanced the conciseness and rigor of the manuscript (lines 103-107).

Revision 13. housed individually!! Please clarify.

Response 13: We are especially grateful for the reviewers' suggestions. We apologize for any unclear phrasing and have incorporated the revisions into the original manuscript. Once again, we sincerely thank the reviewers for enhancing the quality of this manuscript. (Lines115)

Revision 14. ad libitum!!! Please clarify.

Response 14: We sincerely appreciate the reviewer's suggestions. We deeply apologize for the inaccuracies in the original wording and have made the necessary revisions. Once again, we thank the reviewer for their meticulous attention and valuable contributions to enhancing the quality of this manuscript. (Line 116)

Revision 15. In Table 1, the calcium level is higher in the treatment group. Don’t you think this could influence the results?

Response 15: Thank you for your suggestion; I fully agree. We fully recognize that these are not “single-factor” variables, which is a limitation of the current experiment. Future trial designs will specifically address such confounding factors. The use of calcium nitrate in this trial reduces cultivation costs while facilitating operational implementation. Furthermore, existing literature (Li et al., Reference 10) indicates that animals receiving 3% calcium nitrate as a fermentable nitrogen source exhibit comparable growth performance to those in the 1.5% urea group. We once again thank the reviewer for their suggestions, which enhance the logical rigor of the trial. (Line 126)

Revision 16. Is there a specific position in the pens allowed to collect all fecal output?

Response 16: We are particularly grateful for the reviewers' suggestions. We fully agree with these recommendations and have incorporated specific details regarding the fecal collection sites into the manuscript. Once again, we sincerely thank the reviewers for enhancing the quality of this manuscript. (Lines137-138)

Revision 17. How did you collect the rumen fluid? Tube!!!

Response 17: We are particularly grateful for the reviewers' suggestions. We fully agree with these recommendations and have incorporated the specific details of the rumen sampling apparatus into the manuscript. Once again, we sincerely thank the reviewers for enhancing the quality of this manuscript. (Lines 147-148)

Revision 18. Could you please explain the calibration procedure and recovery rate validation? Did you adapt the animals to the chambers?.

Response 18: We sincerely appreciate the reviewers' suggestions and fully agree with these comments. We have revised the text accordingly to explicitly state that goats underwent a 15-day adaptation period in the test chamber. Calibration: Perform “zero and span calibration” of the sensor using standard gases to ensure accurate readings. Recovery rate: A known volume of CH₄/CO₂ mixed gas was injected into the compartment to determine the amount the system could “recover.” A recovery rate within the range of 90–110% is considered acceptable. All other specific operational steps followed the experimental protocol described by Wang et al. (Reference 13). We once again thank the reviewers for their contributions to enhancing the quality of this paper. (Lines155-156)

Revision 19. replace “dry matter” with “DM”.

Response 11: We sincerely appreciate the reviewer's suggestions and apologize for the oversight. We have corrected the DM. Once again, we thank the reviewer for their contributions to enhancing the quality of this manuscript. (Line 172)

Revision 20. replace “crude protein” with “CP”.

Response 20: We sincerely appreciate the reviewer's suggestions and deeply apologize for any oversights. The revisions have been incorporated into the manuscript. We extend our gratitude once again for the reviewer's contributions to enhancing the quality of this paper. ( Line172)

Revision 21. This section is not really clear to the readers. Please rewrite.

Response 21: We appreciate the reviewers' detailed statistical comments and acknowledge the potential limitations in our analytical approach. These statistical limitations and their potential impact on the interpretation of results have been addressed. We have made every effort to revise the manuscript accordingly. However, due to the limited number of replicates within each stage, we introduced a time covariate to account for potential temporal trends. We recognize that linear models may inadequately capture stage-specific variability when using only two time points; for most parameters, we employed independent samples t-tests for straightforward between-group comparisons. We acknowledge this approach does not explicitly account for stage-specific or repeated measures structures. For rumen fermentation parameters (measured pre-feeding and 3 hours post-feeding), we now explicitly state these constitute repeated measures data. Although small sample sizes precluded more complex mixed-model analyses, we have supplemented the text to confirm assumptions regarding normality, homogeneity of variance, and the presence of outliers. We will rigorously consider these factors in future trial designs and again thank the reviewers for their suggestions to enhance the manuscript's scientific rigor. (Lines223-244)

Revision 22. Rename this Table to be “Table 2.” Also, other tables need to be labeled correctly.

Response 22: Thank you for pointing this out. We have now re-numbered all data tables throughout the manuscript sequentially as Table 1, Table 2, Table 3, etc., and cross-checked every in-text citation, figure legend, and appendix reference to ensure complete consistency. We sincerely thank the reviewers for identifying this fundamental and critical issue and apologize for any inconvenience caused during the review process(Lines262-274)

Revision 23. Results of ADG and DMI were similar, so how do you explain the large CO2 reduction since it's linked to metabolic rate?

Response 23: We sincerely appreciate the reviewer's suggestions and fully concur with their considerations. We also recognize that the reduction in carbon dioxide, which does not participate in metabolism, may be attributed to calcium nitrate promoting ruminal propionic acid production, thereby decreasing carbon dioxide yield. We extend our gratitude once again to the reviewer for their contributions to enhancing the quality of this manuscript.. (Lines268-269)

Revision 24. In this Table, SEM is too low for ADG. Please recheck.

Response 24: We sincerely appreciate the reviewer's suggestions and apologize for the error. We have corrected the data accordingly. We once again thank the reviewer for their contribution to enhancing the rigor of this manuscript. (Line 290)

Revision 25. nitrogen balance data did not differ between the treatments. However, the abstract and introduction imply improved N utilization. This is not consistent..

Response 25: Thank you for your valuable feedback. We have carefully reviewed the statements regarding nitrogen utilization efficiency in the conclusion section and have accordingly rewritten and deleted certain passages. The revised text now strictly reflects the statistically non-significant results reported in the paper, thereby avoiding any overinterpretation. We sincerely appreciate the reviewer's rigorous scrutiny, which has helped us enhance the objectivity and accuracy of our conclusions. (Lines 295-299)

Revision 26. The results indicate that Nh3-N is decreased, but in the discussion (lines 480-484), the authors state it is increased. Please clarify this contradiction.

Response 26: We appreciate the reviewer's suggestions. We fully agree with your perspective and sincerely apologize for the oversight. We have now verified the ammonia nitrogen data in the abstract, results, and discussion sections. The results indicate an increase, though no significant difference was observed. We are deeply grateful for the reviewer's rigorous scrutiny, which has helped us enhance the objectivity and accuracy of our conclusions. (Lines 480-484)

Revision 27. This statement is not correct: “may enhance….utilization” Please fix it.

Response 27: We sincerely appreciate the reviewer's suggestions. We deeply apologize for any inaccuracies in the original manuscript and have made the necessary revisions. Once again, we thank the reviewer for their meticulous attention and valuable contributions to enhancing the quality of this manuscript. Special thanks to the reviewer for their meticulous review and valuable suggestions, which have significantly enhanced the academic rigor and quality of the original manuscript. (Line 541)

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor and Authors,

The manuscript addresses a relevant and timely research question on methane mitigation through calcium nitrate supplementation in goats. However, fundamental methodological and statistical concerns persist after the first round of revision. I recommend once more major revision.

Best regards.

Major Comments:

Authors acknowledged the limitation and cited literature supporting the safety of the dose used (19.4 g/kg DM, below 30 g/kg threshold). While the literature justification is reasonable, the absence of MetHb data remains a significant limitation. The authors should explicitly state this as a study limitation in the Discussion section with stronger language than currently provided. The claim that the dose was ‘35% below the toxicity threshold’ is somewhat misleading, as individual animal variation and breed-specific responses could narrow this margin.

6 animals per treatment, individual as experimental unit, two consecutive batches due to pen limitations.The clarification is helpful but raises a new statistical concern: if batches were run at different times, batch is a systematic factor that must be included in the statistical model (as a fixed or random effect). The current analysis does not properly account for this batch structure. With only n=3 per batch, any batch-to-batch variation could substantially bias results. This remains a major concern.

Authors acknowledged confounding but justified it as reflecting ‘practical farm conditions’ and cited Li et al. showing similar performance. This response is scientifically unsatisfactory. A controlled experiment published in a peer-reviewed journal must isolate the variable of interest. Justifying confounding as ‘practical’ undermines the internal validity of the study. The Ca difference (0.31% vs 0.81%) alone could affect rumen buffering capacity and microbial ecology. The SBM change (11.35% vs 7.55%) alters amino acid profiles, rumen-degradable protein, and fermentation patterns independently of nitrate effects. This remains a fundemental design flaw that limits the conclusions that can be drawn.

 

Added Shapiro–Wilk, Levene’s test, and visual inspection statements. Acknowledged small sample limitation for mixed models. Adding assumption-checking statements is a step forward, but the fundamental statistical concerns remain: The batch/phase structure is not accounted for in any model. Pre-feeding and 3h post-feeding VFA measurements on the same animals are repeated measures and require appropriate modeling. Simply stating that sample size precluded mixed models is not acceptable-even a simple paired analysis or split-plot approach would be more appropriate than independent t-tests.

 Authors stated original values were incorrect and provided corrected values (CON: 49.461, CAL: 39.828, p = 0.283).The corrected values now show the expected direction (CON > CAL), but the difference remains non-significant (p = 0.283). This non-significant metric should be transparently discussed.  

Revised conclusions to reflect non-significant results more accurately.  The conclusions have been toned down, but the Simple Summary (L13-24) still contains language suggesting calcium nitrate is ‘effective in diminishing methane emissions, improving digestion, and enhancing the microbial environment.’ The ‘improving digestion’ claim is not supported, as nutrient digestibility was numerically lower in the CAL group (Table 3). This must be corrected.

L51: ‘including cow, sheep and goats’ should be ‘including cattle, sheep, and goats.’ ‘Cow’ refers to adult females only.

L87: ‘We assume that adding calcium nitrate will not adversely affect...’ — Use ‘We hypothesized’ rather than ‘assume’ in scientific writing.

L106: ‘Each sheep is housed’ — Should be ‘Each goat was housed.’ Also, there is a missing space before ‘They received.’

L137: ‘The participants’ average daily weight gain’ — ‘Participants’ is used for human subjects. Use ‘animals’ or ‘goats.’

L191-192:  ‘Samples of rumen content were obtained from six black goats in each experimental group.’ — This contradicts the design where only 3 goats per group were sampled per phase. Please clarify whether all 6 goats were sequenced and how samples from the two batches were handled bioinformatically.

L365-367: The LEfSe results appear to conflict with the phylum/genus-level findings. The LEfSe analysis identifies Cyanobacteria and Acetitomaculum as enriched in the CAL group, yet Table 6 shows Cyanobacteria was numerically lowerin CAL (0.062 vs 1.199), and Acetitomaculum was numerically lower in CAL in Table 7 (4.276 vs 7.146). Please reconcile these contradictions.

 

Author Response

Dear reviewer,

We are very grateful to Reviewer for reviewing the paper so carefully.

We have tried our best to improve the manuscript and have modified some. Specific changes are as follows:

Re: Manuscript ID: animals-4184893 and Title: Effect of dietary calcium nitrate addition on methane emission, nitrogen excretion, and ruminal fermentation parameters and microbiota in Liuyang black goats.

Comments and Suggestions for Authors

Revision 1. Authors acknowledged the limitation and cited literature supporting the safety of the dose used (19.4 g/kg DM, below 30 g/kg threshold). While the literature justification is reasonable, the absence of MetHb data remains a significant limitation. The authors should explicitly state this as a study limitation in the Discussion section with stronger language than currently provided. The claim that the dose was ‘35% below the toxicity threshold’ is somewhat misleading, as individual animal variation and breed-specific responses could narrow this margin.

Response 1: We sincerely thank the reviewers for pointing out this critical issue. We deeply apologize for failing to clearly articulate the differences in safety thresholds across species regarding safe doses in the original manuscript, as well as for failing to adequately address the limitations arising from the lack of methemoglobin data. We have provided additional clarification in the “Discussion” section and made the necessary revisions. We sincerely apologize for this oversight in the original manuscript and have systematically revised and supplemented the main text in accordance with your recommendations. We thank the reviewers for their rigorous review, which will help enhance the rigor and quality of the paper. (lines 425–427).

Revision 2. 6 animals per treatment, individual as experimental unit, two consecutive batches due to pen limitations. The clarification is helpful but raises a new statistical concern: if batches were run at different times, batch is a systematic factor that must be included in the statistical model (as a fixed or random effect). The current analysis does not properly account for this batch structure. With only n=3 per batch, any batch-to-batch variation could substantially bias results. This remains a major concern.

Response 2: We sincerely thank the reviewer for pointing out this critical issue and deeply apologize for the lack of clarity in our previous description. The experiment was conducted in two cycles primarily to increase the sample size; it was repeated twice, with three sheep selected from each group for the first cycle and the remaining three used for the second cycle. In designing the experiment, we took into account both the practical limitations of the metabolic chamber and the need for sufficient sample size. This experiment has been replicated to validate the results. In our previous statistical analysis, we treated time as a covariate and included it in the statistical model: Yi= μ + Ti +βt⋅tj + εi. In this context, Yi represents the average treatment value observed for the i-th dietary group during the 5-day sampling period. The population mean is denoted by μ. The fixed effects of the two treatments (CON and CAL, corresponding to i = 1 and 2, respectively) are denoted by Ti. The term βt denotes the effect associated with the time phase, indicating how time influences the dependent variable. The time covariate tj is defined such that tj = 1 corresponds to the initial time phase, while tj = 2 denotes the subsequent time phase. Furthermore, when measuring methane and carbon dioxide at each stage, we calibrate the metabolic chamber and calculate the corresponding recovery rates, which are then used to correct the measured values. The calibrated metabolic chamber is used to correct for temporal errors. We sincerely apologize for not explicitly stating in previous versions that this model is intended to analyze growth performance, CH₄ emissions, CO₂ emissions, and apparent nutrient digestibility; this issue has been corrected in the revised manuscript. We have made the necessary revisions based on your suggestions. We appreciate the reviewers’ suggestions, which have enhanced the rigor and quality of this paper. (Lines 222–224).

Revision 3. Authors acknowledged confounding but justified it as reflecting ‘practical farm conditions’ and cited Li et al. showing similar performance. This response is scientifically unsatisfactory. A controlled experiment published in a peer-reviewed journal must isolate the variable of interest. Justifying confounding as ‘practical’ undermines the internal validity of the study. The Ca difference (0.31% vs 0.81%) alone could affect rumen buffering capacity and microbial ecology. The SBM change (11.35% vs 7.55%) alters amino acid profiles, rumen-degradable protein, and fermentation patterns independently of nitrate effects. This remains a fundemental design flaw that limits the conclusions that can be drawn.

Response 3: We sincerely thank the reviewer for pointing out this critical issue and deeply apologize for any inconvenience caused during the review process. We fully understand the reviewer’s concerns. Regarding your comment on the relatively high calcium (Ca) content (0.81), we have reduced the calcium carbonate content to zero in the treatment group’s formulation; the calcium nitrate we added already contains calcium. Given the amount added, the calcium ion content here will not affect our experimental results. Furthermore, regarding the differences in amino acid composition resulting from the lower soybean meal content in the experimental group, calcium nitrate, as a non-protein nitrogen source, can effectively substitute for protein. The ammonia nitrogen produced during its degradation can be utilized by microorganisms for the synthesis of bacterial proteins, essential amino acids, and non-essential amino acids. Furthermore, a study by Li et al. found that the production performance of sheep fed 3% calcium nitrate as a fermentable nitrogen source was comparable to that of sheep fed 1.5% urea as a fermentable nitrogen source (Reference 10). Therefore, this will not affect our experimental results. In future studies, we will place greater emphasis on experimental design. We appreciate the reviewers’ suggestions, which will help improve the quality and rigor of our paper.

Revision 4. Added Shapiro–Wilk, Levene’s test, and visual inspection statements. Acknowledged small sample limitation for mixed models. Adding assumption-checking statements is a step forward, but the fundamental statistical concerns remain: The batch/phase structure is not accounted for in any model. Pre-feeding and 3h post-feeding VFA measurements on the same animals are repeated measures and require appropriate modeling. Simply stating that sample size precluded mixed models is not acceptable-even a simple paired analysis or split-plot approach would be more appropriate than independent t-tests.

Response 4: We sincerely thank the reviewers for pointing out this critical issue and apologize for any inconvenience caused during the review process. The experiment was conducted in two phases to increase the sample size; this was due to practical limitations on the availability of the metabolic chamber. Each experiment was repeated twice to improve the accuracy of the results. Considering the effects of time and batch composition, we treated time as a covariate and analyzed the data on growth performance, CH₄ emissions, CO₂ emissions, and apparent nutrient digestibility using the model Yi = μ + Ti + βt·tj + εi. We calibrated the metabolic chamber at the beginning and end of each phase and calculated recovery rates to correct the measurement results. Additionally, for samples taken from the same animal, volatile fatty acid (VFA) data collected 3 hours before and 3 hours after feeding constituted replicate measurements. These data were used to elucidate the rate at which calcium nitrate releases ammonia nitrogen in the rumen and to verify whether calcium nitrate causes ammonia toxicity and nitrogen wastage. It is essential to examine the effects of calcium nitrate on changes in rumen fermentation parameters immediately upon entering the rumen. We deeply apologize for this omission and have made the necessary revisions based on your suggestion (lines 95–100).

Revision 5. Authors stated original values were incorrect and provided corrected values (CON: 49.461, CAL: 39.828, p = 0.283).The corrected values now show the expected direction (CON > CAL), but the difference remains non-significant (p = 0.283). This non-significant metric should be transparently discussed.

Response 5: We are particularly grateful for the reviewers’ suggestions. We fully agree with your comments and have made revisions accordingly. We would like to once again express our sincere gratitude to the reviewers for helping to improve the quality of this manuscript (Lines 424–425).

Revision 6. Revised conclusions to reflect non-significant results more accurately.  The conclusions have been toned down, but the Simple Summary (L13-24) still contains language suggesting calcium nitrate is ‘effective in diminishing methane emissions, improving digestion, and enhancing the microbial environment.’ The ‘improving digestion’ claim is not supported, as nutrient digestibility was numerically lower in the CAL group (Table 3). This must be corrected.

Response 6: We are particularly grateful for the reviewer’s suggestions. We fully agree with your comments and have revised the summary accordingly. We would like to once again express our sincere gratitude to the reviewer for helping to improve the quality of this manuscript. (Lines 21–23).

Revision 7. including cow, sheep and goats’ should be ‘including cattle, sheep, and goats.’ ‘Cow’ refers to adult females only.

Response 7: We are particularly grateful for the reviewers’ suggestions. We fully agree with your comments and have revised the manuscript accordingly. We would like to once again express our sincere gratitude to the reviewers for helping to improve the quality of this manuscript. (Line 51).

Revision 8. We assume that adding calcium nitrate will not adversely affect...’ — Use ‘We hypothesized’ rather than ‘assume’ in scientific writing.

Response 8: We are particularly grateful for the reviewers’ suggestions. We fully agree with your comments and have revised the manuscript accordingly. We would like to once again express our sincere gratitude to the reviewers for helping to improve the quality of this manuscript. (Line 87).

Revision 9. Each sheep is housed’ — Should be ‘Each goat was housed.’ Also, there is a missing space before ‘They received.

Response 9: We are particularly grateful for the reviewers’ suggestions. We fully agree with your comments and have revised the manuscript accordingly. We would like to once again express our sincere gratitude to the reviewers for helping to improve the quality of this manuscript. (Line 106).

Revision 10. The participants’ average daily weight gain’ — ‘Participants’ is used for human subjects. Use ‘animals’ or ‘goats.

Response 10: We are particularly grateful for the reviewers’ suggestions. We fully agree with your comments and have revised the manuscript accordingly. We would like to once again express our sincere gratitude to the reviewers for helping to improve the quality of this manuscript (Line 137).

Revision 11. Samples of rumen content were obtained from six black goats in each experimental group.’ — This contradicts the design where only 3 goats per group were sampled per phase. Please clarify whether all 6 goats were sequenced and how samples from the two batches were handled bioinformatically.

Response 11: We sincerely thank the reviewers for pointing out this critical issue and apologize for the lack of clarity in the “Materials and Methods” section during the review process. We have revised the original text as follows: “Each group consisted of 6 goats with an average weight of 28 ± 0.2 kg; each goat served as an independent replicate. The experiment was divided into two 20-day cycles, totaling 40 days. Feeding conditions remained consistent throughout the entire experiment. During the first 20 days, samples were collected from 3 goats in each group; during the second 20 days, samples were collected from the remaining 3 goats in each group.” All 6 goats were sequenced. This revision has been applied to lines 95–100. We deeply apologize for this oversight and once again thank the reviewer for their suggestions, which have helped improve the quality of the manuscript (lines 95–100).

Revision 12. The LEfSe results appear to conflict with the phylum/genus-level findings. The LEfSe analysis identifies Cyanobacteria and Acetitomaculum as enriched in the CAL group, yet Table 6 shows Cyanobacteria was numerically lowerin CAL (0.062 vs 1.199), and Acetitomaculum was numerically lower in CAL in Table 7 (4.276 vs 7.146). Please reconcile these contradictions.
Response 12: We sincerely thank the reviewers for pointing out this critical issue and deeply apologize for the inaccuracies in the description of the results in the original manuscript. We have made the necessary revisions to the “Results” section. We deeply regret this oversight and once again thank the reviewer for their suggestions, which have helped improve the quality of the manuscript. We would like to extend our special thanks to the reviewer for their meticulous review and valuable suggestions, which have significantly enhanced the academic rigor and quality of the original manuscript. (Lines 634–637).

 

Reviewer 2 Report

Comments and Suggestions for Authors

The author has carefully revised and checked according to the comments.

Author Response

Dear reviewer,

We are very grateful to Reviewer for reviewing the paper so carefully.

We have tried our best to improve the manuscript and have modified some. Specific changes are as follows:

Re: Manuscript ID: animals-4184893 and Title: Effect of dietary calcium nitrate addition on methane emission, nitrogen excretion, and ruminal fermentation parameters and microbiota in Liuyang black goats.

Comments and Suggestions for Authors

We sincerely appreciate your constructive comments and suggestions. They have been extremely helpful in improving the quality and rigor of our paper. Once again, thank you for your professional review and hard work; this is a great encouragement to our research.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors addressed all comments in the previous revision; I have no reason not to accept the manuscript in its current form. Thanks 

Author Response

Dear reviewer,

We are very grateful to Reviewer for reviewing the paper so carefully.

We have tried our best to improve the manuscript and have modified some. Specific changes are as follows:

Re: Manuscript ID: animals-4184893 and Title: Effect of dietary calcium nitrate addition on methane emission, nitrogen excretion, and ruminal fermentation parameters and microbiota in Liuyang black goats.

Comments and Suggestions for Authors

We sincerely appreciate your constructive comments and suggestions. They have been extremely helpful in improving the quality and rigor of our paper. Once again, thank you for your professional review and hard work; this is a great encouragement to our research.