Effects of Rumen-Protected Lysine and Methionine Supplementation on Lactation Performance in Holstein Dairy Cows: A Meta-Analysis
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsAs acknowledged by the authors, several meta-analyses have previously evaluated the effects of supplementing dairy cattle diets with rumen-protected amino acids, particularly rumen-protected methionine. In this context, the authors’ effort to broaden the scope of the analysis is noteworthy, as it includes not only coated amino acid products but also methionine analogs, whether administered directly or in forms chemically linked to isopropyl alcohol. Additionally, the authors attempt to assess the impact of rumen-protected methionine supplementation under heat stress conditions, while appropriately recognizing the limited availability of published data in this specific area.
However, as with many previously published meta-analyses, a fundamental underlying issue is not adequately addressed. Specifically, the analysis assumes as accurate the bioavailability values reported by manufacturers for each amino acid source included. Although the authors acknowledge that “rumen protection efficiency” may differ among products, the statistical comparisons rely heavily on manufacturer-reported estimates. This approach introduces a potential source of bias that may affect the interpretation of the results.
It is well established that different companies employ distinct coating technologies, which can vary substantially in their effectiveness. This variability is particularly pronounced among methionine analog products. Furthermore, there is no universally accepted or standardized method for determining amino acid bioavailability. As a result, companies utilize different methodologies to assess product efficacy, leading to inconsistencies in reported values. In practice, bioavailability claims are largely determined by the manufacturer, further compounding the potential for bias.
Importantly, when independent evaluations are conducted using a common methodology across different products, the resulting bioavailability estimates can differ markedly from manufacturer-reported values and from one product to another. Therefore, conducting a meta-analysis that compares products based on bioavailability values derived from disparate methodologies may, at best, underestimate the true biological responses in dairy cows when the intended delivery of the amino acid was less (due to an overestimation of the efficiency of the coating) but still there was a response. Or worse, when the responses are not as high due to assuming delivery of the subject amino acid and in fact, there was less than the desired amount, which can lead to misleading conclusions.
For these reasons, I strongly encourage the authors to more explicitly address this limitation. A more critical evaluation of bioavailability assumptions—and their potential impact on the analysis—would strengthen the manuscript and better differentiate it from previous meta-analyses in which this issue has been largely overlooked.
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
Your manuscript entitled "Effects of Rumen-Protected Lysine and Methionine Supplementation on Lactation Performance in Dairy Cows: A Meta-Analysis" might be suitable for publication in Animals after major revision. Please, see below a list of comments/suggestions to be applied by you before accepting it to be published.
Yours sincerely,
Reviewer.
L14-L42 Try to reformulate your Simple Summary and Abstract to provide relevant scientific knowledge about your topic by avoiding to give simple information.
L86-95 Delete this paragraph due to in your research only 3 references to heat stress were found and with them there is not enough evidence to support your assumptions.
L106-L115 Try to be consistent in your aims to avoid any misinterpretation. If only 3 articles were found on heat-stress condictions, you should consider to not focus on that.
Table 1: It might be included in a Supplementary file and a short explanation given in the text.
L194-232 Further explanation is needed in Statistical analysis to describe the statistical tests perfomed, the factors under study, the model tested, etc.
Table 2: Heat stress references should be deleted due to if only 3 articles were found by the authors there is not enough support evidence to include them in this manuscript. Verify that the other 42 references related to non-heat stress are present and cited.
L248 Replace "45 studies" by "42 studies".
L236-L249 Summarize it. Try to avoid any repetitionof what is shown in Figure 1.
Table 3: It might be included in a Supplementary file and a short explanation given in the text.
Figure 3: It might be deleted due to only 3 references support the data presented herein.
Check hyphoning throughout the manuscript.
L484-L502 Delete this paragraph due to in your research only 3 references to heat stress were found and with them there is not enough evidence to support your assumptions.
L553-L560 Rewrite your Conclusions to avoid any repetition of results and to mention heat stress due to only 3 articles were found. Try to provide new ideas for future research studies and explain the implications of your finding for the future of the dairy sector.
L569-L763 Check your references. Please, follow Animals' instructions for correct citation.
Author Response
We have revised the content accordingly. Please kindly refer to the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsGeneral comments:
This meta-analysis aimed to evaluate the effect of limiting amino acids (lysine and methionine ) on milk yield and milk components of Holstein dairy cows under heat stress and non-heat stress (or unknown). This is an interesting approach. The background clearly identifies gaps and inconsistencies in the literature to justify the objectives of the study. Apparently, the scientific soundness of this study is mainly due to variables with the greatest differences and low heterogeneity, as mentioned by the authors in the conclusions/abstract. There are relevant doubts regarding the use of exploratory regression analysis, even if considered as “exploratory” data (please see the specific comments). There are not performed at the cow level, most are not significant or very low, the determination coefficients are for linear models, but non-linear models are also presented, someones present a tendency, etc. Consequently, the authors use (erroneously) terms such as linear, non-linear, numerical, trends, etc., which have a specific statistical definition. In my opinion, we cannot retain an adequate conclusion from this analysis. Even if we consider it acceptable, only moderate determination coefficients should be retained in the figures. Nonetheless, in my opinion, the data reported in Figs. 2 and 3 can justify the new insights of this study. Strengths and limitations of the study should be clearly reported.
Specific comments:
I suggest adding “Holstein” to the title.
L193, 306: Usually, the Table and Figure numbers appear sequentially. This can reduce the readability of your study.
L251: Please use capital letters in points 2 to 5.
L306-314: There are some doubts in the exploratory regression analysis: the sample size (samples/cows) was not reported (even though this limitation is mentioned); the determination coefficients are low; the p-values are not significant (suggesting that the sample sizes are small)- there are no trends (L306), except for fig. 4C ; the blue lines seem to represent polynomial correlations. Will be difficult to obtain conclusions from this data.
L333-341: The same issue for Fig. 5.
L362: There are trends for Fig. 6A and 6C.
L393, 344: As other figures, Fig. 7 and 8 and the respective text are based on a very low determination coefficient for a low sample size and high p-values: the question is whether we can consider these results as a preliminary form (exploratory). Probably, these analyses should be performed at the animal (or group) level.
Author Response
We have revised the content accordingly. Please kindly refer to the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsDear Authors,
It's a pleasure for me to inform you that your manuscript has been improved according to the suggestions provided and it might be now suitable for publication in Animals in present form.
Congratulations to all of you for this achievement!
Looking forward to hearing from you in another occasion.
Yours sincerely,
Reviewer.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
Thank you for providing this revised version. The suggestions and comments of this reviewer were taken into consideration. Furthermore, the substantial and cautious interpretation of the results was adequately supported by the meta-analysis and published literature.
A mention of Supplementary Figures 5-12 should be inserted into the text.
