Review Reports - Milk Fatty Acid Profiling as a Tool for Estimating Methane Emissions in Conventionally Fed Dairy Cows

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The abstract must include the research objective or research hypothesis. Lines 28-32 should be changed, the information is repeated.
In the introduction, you can write briefly about the factors that determine the chemical composition of milk and then describe the role of methane. The introduction needs to be changed, especially lines 71-86 (the description is too general). The undertaking of research requires justification.
Materials and Methods: Please add: how many cows in each group were first-calf heifers and how many were multiparous cows. At what time of year were the studies conducted? The fatty acid content is influenced by the length of the day (and especially by melatonin signals). What was the temperature in the building where the cows were kept?. When were the milk samples collected (morning or evening milking)? Were the samples collected from the entire udder or from a specific individual quarter?. What was the weight and condition of the cows before the experiment began?
Results well described, substantively and clearly presented in tables.
The discussion needs to be rewritten. It should describe what the research specifically demonstrated. The description is extensive and general, and much of the information is repeated several times. It should describe what new findings the research revealed and whether they can be applied in practice.
Summary: repeated information from the discussion, necessary rewriting and description of the application value of the research
The literature is well chosen, and it is worth noting that there is not much literature available on this subject. The studies are interesting and provide new information.

Comments for author File: Comments.pdf

Author Response

AU: Thank you for your time, thoughtful feedback, and constructive suggestions, which have significantly improved the quality and clarity of the manuscript. We greatly appreciate your time, effort, and expertise.

The abstract must include the research objective or research hypothesis. Lines 28-32 should be changed, the information is repeated.

AU: We revised the abstract to explicitly state the research objectives (L16-19). In addition, we refined the wording in L28-31 to eliminate redundancy and improve clarity.

In the introduction, you can write briefly about the factors that determine the chemical composition of milk and then describe the role of methane. The introduction needs to be changed, especially lines 71-86 (the description is too general). The undertaking of research requires justification.

AU: We revised the introduction accordingly (see L46-81).

Materials and Methods: Please add: how many cows in each group were first-calf heifers and how many were multiparous cows. At what time of year were the studies conducted? The fatty acid content is influenced by the length of the day (and especially by melatonin signals). What was the temperature in the building where the cows were kept?. When were the milk samples collected (morning or evening milking)? Were the samples collected from the entire udder or from a specific individual quarter?. What was the weight and condition of the cows before the experiment began?

AU: We did not have groups in our experimental design. All 12 primiparous cows received a TMR specifically formulated for first-calf heifers, while multiparous cows received a diet tailored to their milk yield (i.e., low- or high-yield diet). For clarity we included the number of cows on each diet (see L93-94). We added the time of year and the average temperature in the barn for the study period (see L87 and 90-91). We acknowledge that the photoperiod affects melatonin secretion, however, all cows included in this study were housed in a tie-stall barn with artificial lighting which likely limited seasonal photoperiod effects. The number and timing of milkings are reported in L99-100 of the original manuscript, though we modified this statement in the revised version to improve clarity (see L95). In addition, we expanded the description of the in-line milk sampling system (see L118-120), which collects a representative composite sample from all four quarters during each milking via connection to the main collection line of the milking unit. Body weight and condition scores of cows are reported in Table 3. These measurements were only collected once at the beginning of the trial as the observation period was short. We included additional table subtext to clarify this point (see L209).

The discussion needs to be rewritten. It should describe what the research specifically demonstrated. The description is extensive and general, and much of the information is repeated several times. It should describe what new findings the research revealed and whether they can be applied in practice.

AU: We revised the discussion to emphasize the new findings of our research. Redundant or overly general statements were removed (see L307-384).

Summary: repeated information from the discussion, necessary rewriting and description of the application value of the research

AU: The revised version now demonstrates the key outcomes and their relevance to CH4 mitigation and dairy management (L386-394).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study aimed to identify milk fatty acids that could serve as biomarkers for enteric CH4 emissions in lactating dairy cows fed an industrial standard diet and to develop models that accurately estimate CH4 output (g/d), yield (g/kg DMI), and intensity (g/kg ECM). Mitigation of methane (CH4) emissions on dairy farms is necessary to reduce the impact of dairy production on climate change.This topic is relevant and significant, and the relationship between the composition of milk fatty acids and enteric methanogenesis is well known. The results of the study make some contribution to solving the problem of using the milk fatty acid profileas an indirect method for predicting CH4 emissions from dairy cattle.

The manuscript is written in clear and professional English, and its general structure follows the standard scientific format. The authors provide a good literary base to support the research. The research hypothesis and goals are clearly formulated. The presented data is generally consistent with the tasks set.

The study presents analyses of the fatty acid profiles of cow's milk to identify potential biomarkers for predicting CH4 release and to develop models that accurately estimate CH4 output, yield, and intensity. The experimental design is adequate, but some methodological details require a detail description, especially regarding the extraction of lipids, the preparation of methyl esters and FAME correction for mass discrepancy. In addition, the identification of fatty acids only by comparison of retention times with known FAME standards is insufficient and inconclusive. Moreover, three exotic fatty acids (11-cyclohexyl-11:0 and 13:0-anteiso, as well as 20:3c5,c8,c11), which had not previously been described in cow milk, were identified in the present study.These fatty acids show a moderate or strong positive correlation with CH4 values. These fatty acids were used as biomarkers for enteric CH4 emissions in lactating Holstein dairy cows fed an industrial standard diet, because they exhibiting moderate to strong positive correlations with CH4 metrics.To confirm the identification of fatty acids, additional analysis methods should be applied, for example, such as GC-MS of methyl esters. Perhaps the preparation of butyl esters of fatty acids would be more effective.

The study is devoted to an relevant environmental topic, but its novelty and contribution to the existing literature are limited, since similar experiments on the use of milk fatty acid profiles as an indirect method for predicting CH4 emissions from dairy cattle have been conducted previously.

The main conclusion of the manuscript "Collectively, these findings demonstrate that the milk FA profile provides a biologically relevant and reliable foundation for predicting CH4 emissions." is obvious from previous studies. The "Conclusions" section in its current version is rather an extension of the "Discussion" section. It should be reviewed and changed to focus on the novelty of the results obtained.

Author Response

AU: Thank you for your time, effort, and constructive suggestions, which have significantly improved the quality of this manuscript. We sincerely appreciate your time, expertise, and feedback.

The study presents analyses of the fatty acid profiles of cow's milk to identify potential biomarkers for predicting CH4 release and to develop models that accurately estimate CH4 output, yield, and intensity. The experimental design is adequate, but some methodological details require a detail description, especially regarding the extraction of lipids, the preparation of methyl esters and FAME correction for mass discrepancy. In addition, the identification of fatty acids only by comparison of retention times with known FAME standards is insufficient and inconclusive. Moreover, three exotic fatty acids (11-cyclohexyl-11:0 and 13:0-anteiso, as well as 20:3c5,c8,c11), which had not previously been described in cow milk, were identified in the present study. These fatty acids show a moderate or strong positive correlation with CH4 values. These fatty acids were used as biomarkers for enteric CH4 emissions in lactating Holstein dairy cows fed an industrial standard diet, because they exhibiting moderate to strong positive correlations with CH4 metrics. To confirm the identification of fatty acids, additional analysis methods should be applied, for example, such as GC-MS of methyl esters. Perhaps the preparation of butyl esters of fatty acids would be more effective.

AU: We appreciate your suggestion to elaborate on the lipid extraction and FAME preparation procedures. While we did not describe our lipid extraction and FAME preparation in detail in this work, references are provided to previous work detailing the methodology (see L143-145). The two works regarding FAME preparation and GC-FID analysis (Bainbridge et al., 2015 [https://doi.org/10.1021/acs.jafc.5b00857] and Unger et al., 2020 [https://doi.org/10.1021/acs.jafc.9b07390]) are works published previously by our lab. Given that our procedures followed these protocols, we felt additional methodological detail was not necessary to avoid redundancy. 11-cyclohexyl-11:0, 13:0-anteiso, and 20:3 c5,c8,c11 have previously been reported in the literature (see L340-341). We included further explanation of our identification protocol to our methods (see L150-157). FAME identification via GC-MS was not used in this particular study because we were interested in positional isomers (e.g., 18:1 fatty acids) and branched-chain isomers (e.g., 13:0-anteiso) which cannot be reliably resolved via GC-MS (Van Pelt and Brenna, 1999 [https://doi.org/10.1021/ac981387f]; Ran-Ressler et al., 2012 [https://doi.org/10.1194/jlr.D020651].

AU: We acknowledge that the relationship between milk fatty acid profiles and methane emissions has been explored in previous studies. However, our work introduces a new perspective by focusing on cows fed standard industry diets rather than those with experimental formulations and supplements. We revised the Introduction to emphasize this point and to more clearly articulate the study’s unique contribution (see L60-74).

AU: The conclusion has been rewritten to clearly highlight the unique aspects of this study (see L386-394).