The Importance of Cow-Individual Effects and Diet, Ambient Temperature, and Horn Status on Delayed Luminescence of Milk from Brown Swiss Dairy Cows

Round 1

Reviewer 1 Report

This paper evaluates the influence of diet, ambient temperature, and horn status on delayed luminescence of Milk from Brown Swiss Dairy Cows.

In general, the paper is addressing an interesting technique, delayed luminescence, and is well organized and written. The main drawback of the study is the weak justification of the study regarding its potential application. In other words, the authors should try to clearly justify why this study is needed, and what would be the specific use of measuring delayed luminescence in milk. In fact, the potential application of the measuring technique is clearer in some of the examples that authors have mentioned in the introduction. For instance, distinguishing between organic and conventional milk, or indicating subacute rumen acidosis, among others. However, it is more difficult identifying the potential use in relation with factors studied in this research: hey or hey + concentrates; horned vs disbudded; and ambient temperature (10 and 25 °C).  Authors have claimed in the objectives statement the interest of evaluating the influence of diet, ambient temperature and horn status on the physiology and performances of dairy cows. In this sense, wouldn't it have made more sense to try to predict, for example, some performance parameters from milk fluorescence spectroscopy measurements? Authors should try justifying why stablishing the relation between the studied factors and fluorescence spectroscopy measurements is needed/advisable and relate all this justification with the objectives of the study, in a more comprehensive manner. This approach will help the reader to understand the potential of delayed luminescence measurements in milk regarding the evaluated independent factors, which is not clear yet. Authors should address this query modifying the introduction and the objective statement accordingly. Definitively, some changes to this regard will also be needed in the conclusions, that should discuss the main meaning, application, and impact of the results obtained.

In addition to this main requirement, some specific queries follow:

The authors are using several terms to refer to the optical measurement performed: “florescence excitation spectroscopy”, “light-induced photon emission” and “delayed luminescence”. It would be advisable to refer the most appropriate one and be consistent on its use.  Fluorescence excitation spectroscopy (FES) is a method to measure delayed luminescence. It seems that delayed luminescence is the basic phenomena, which should be, in addition, clearly differentiated from regular fluorescence. Light-induced photon emission is in my opinion a more general term that might be more ambiguous. In this regard, title should also be shorter and more specific. It contains two of these terms.

There are several citations to unpublished materials, but they should be properly cited, following the authors directions (both text and reference list). Lines 61, 312, 322. Please check and correct.

According to the SI units system, a space is used between the number and the symbol to which it refers. This also included °C: x °C rather than x°C. For instance, in line 116 change “45min. in the” to “45 min in the”. Please, change everywhere.

Regarding the experimental design, although the design used seems adequate, changes in milk composition, even from the same animal under identical conditions (one specific treatment), should be expected to affect fluorescence phenomena. Authors should address this issue.

Line 116. Room temperature is typically around 20 °C. Volumetric material used to be calibrated at this temperature. Authors referred room temperature as 15°C. Please indicate why this temperature was used rather than 20 °C.  

Line 121. “Before each cycle, a reference-sample is measured. Authors should specify what was used as a reference and the reason for it.

Lines 135-136. Please, indicate how the emission parameters were derived from the row data.

Regarding the method for FES measurements, indications regarding the measurement conditions should be more detailed. For instance, complete reference of the equipment used, characteristics of the halogen lamp used, including the power or voltage. Other important information is missing such as cuvette characteristics, measurement angle.  Complete reference of filters and photomultiplier used should be provided. A typical delayed fluorescence spectrum would help also the non-familiar reader.  

Please, refer table 1 before presenting it.

Authors should explain why significance was selected using p<0.1 and tendencies defined according to 0.3<p<0.1. Typically, significance would be assumed for p<0.05 and tendencies for 0.1<p<0.05.

Line 173. Regression models should be specified. Additionally, in table 4, total and error DF should be included. Was an intercept used? Regression coefficients and standard error of estimate for coefficients should b reported.  

Lines 185-187. Model quality should also be evaluated by SEP and CV of the regression models.

Table 2. CV should be reported as %.

Line 202. It is surprising that a R2 = 0.15 would be very significant (p=0.001). Please check.

Line 203. m=49.4. why does m stand for? m is not reported in other CV values.

Regarding references list (pp. 13 and 14), please check reference 5. Et al. is not needed if all the authors are properly listed. Reference 16; It is not necessary to state that the document is a “short communication”.

Author Response

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Author Response File: Author Response.docx

Reviewer 2 Report

I have examined this paper. The study is reasonably well set up and the work could be interesting, but there is one major issue: the authors only do one type of measurement on their samples (FES) and treat this measurement as a complete black box. As a result, the work at present has very little meaning unfortunately. There should be proper correlations to components of milk established to allow any interpretation of the data. The authors aim to relate some parameters measured to e.g., mitochondria, but in doing this take the oversimplistic assumption that what was measured on e.g., a yeast system can be translated to milk, not considering all the other components present in milk and/or whether there are even enough cells to give a decent signal. As a result, unfortunately, the work at present presents only numerical findings which cannot (yet) be translated to something meaningful. I feel the authors have to take this next step and be able to interpret their FES data properly before this can become a meaningful publication.

Hence, in my opinion, the authors should provide more data on the milk samples they analysed, e.g., in terms of composition or other properties they wish to relate their FES data to. At present, they try to pass FES off as an established technique for milk analysis, but it isn’t (or at least with the info they provide it does not appear to be). So, if they want to correlate FES data to mitochondria or anything else in milk, they should provide the data to back this up. At present this is not the case. When reading the experimental design, I imagine more data must have been taken than what is currently presented in the manuscript, so I think these need to be shown, both metabolic parameters of the cows as well as milk composition. Without this, the entire discussion is basically speculative and not supported by any results.  

I would also question the statistics. P<0.1 would in my opinion be fairly ‘liberal’ and 0.3<p<0.1 has limited meaning. Why was p<0.05 not considered?  

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

The aim of the paper submitted to Dairy journal is to use Fluorescence Excitation Spectroscopy to Evaluate the Influence of Diet, Ambient Temperature and Horn Status on Light-In-duced Photon Emission of Milk from Brown Swiss Dairy Cows. The paper is well written and need some corrections indicated below.

Line 18. Please precise the wavelength excitation range.

Line 24. Please precise the wavelength excitation range.

Line 26. All the abreviations need to be precised in the document when it fristly appear.

The end of the introduction needs to be reformulated (from line 86 to line 93).

Line 125. How this was done ? Please precise.

Line 127. add a point at the end of the sentence.

Line 142-143. Please give more precision concerning this line.

The authors did not explain how they obtained 152 samples, please explain this point clearly.

Table 2. I am not sure what is the interest to give the CV of the median, min and max. Please explain.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Authors have addressed the queires properly.

Author Response

o.k.

Reviewer 2 Report

Not a whole lot has changed in the paper other than cosmetic changes, so from that perspective my views have not changed either and most of the original comments in the review still apply. From the response of the authors I see that they are happy to treat the measurements as black box, but if that is the case, they should really do so and not put up a large speculative discussion in the paper. At that point, they should simply say 'we found these correlations and we don't know yet what they mean'

Author Response

Dear Reviewer, (09.06.22)

I see your point of view – and I can understand your objection regarding the comparison of milk matrix with yeast / mitochondria matrix. I changed the last part of the discussion. The whole part “DL and metabolic processes” was replaced by “area of application of DL”.

I took the chance to change the focus of this part, because your remarks made me conscious that it may be of more importance for the readers to see in which general fields of application the DL and the different DL parameters may be used to show effects in milk quality. The scientific basis for detailed metabolic explanation is really weak in the field of milk-related results, and you are right, I should stay at the bottom of results and not think too far abroad.

Thank you for your remark!

The changes in detail:

• The header of the section was changed from “DL and metabolic processes” to “Potential area of application of DL”
• The first section was rewritten – according to the new topic (line 491 – 495).
• The last section of the old version was transferred to line 498 – 514
• In the section of line 515 – 522 some wordings were fitted.
• The sections which related to non-milk-matrix were excluded.
• A further reference (on milk-matrix) was added (line 524).
• The last section (L532, “in conclusion…”) was rephrased, focusing on the generalized area of application.

The conclusions were not changed, because they express – to my view – especially the results and the area of application, and are not based on results from matrix-foreign phenomena.