Changes in Protein Metabolism Indicators in Dairy Cows with Naturally Occurring Mycotoxicosis before and after Administration of a Mycotoxin Deactivator

Round 1

Reviewer 1 Report

GENERAL COMMENTS

This is an interesting manuscript but not entirely original. There are several methodological and experimental design issues that should be discussed in more depth.

SPECIFIC COMMENTS

Lines 27-28: I don't see the results in the summary that allow us to conclude this. Also, talking about the "overall health of cows" is a bit risky and was not part of the study, or at least it was not sufficient and appropriately analyzed.

The explanation of the experimental design is somewhat confusing. As far as I understand, the authors used two dairy farms, one with mycotoxins in the diet was treated with the "deactivator" and another without mycotoxins was left as a control. If I properly understood the experimental design, I have some questions: a) was the presence of mycotoxins periodically evaluated in the diet of both farms? Were the cows included in the study balanced according to the number of lactations, daily milk production, body condition or other possible confounders? If this was not done, were these potential confounders included in the subsequent statistical analysis? The farms were different in terms of daily milk production and milk fat composition, how do the authors think these differences did not affect the treatment results? What was the daily milk production per cow?

One of the farms presented significant health problems during the trial (judging from the data presented). Could this condition affect the results?

Table 2: Assuming the two groups were balanced (which they do not appear to be), the control group was not sampled at the same times as the treated group? You only have one data at the beginning of the trial, so how did the authors ensure that the variations found in the blood parameters of the treated cows are not physiological changes?

Line 243: The evaluation of the health status of the cows was not part of the study, no results are presented, nor were statistical analyzes performed. In fact, the farm that had these symptoms at the beginning of the trial was the Control

Line 252: "Undoubtedly." With the experimental design used, the authors would not seem to be in a position to conclude with such emphasis, since two different farms were used and it was not (apparently) corrected for possible confounding factors.

Author Response

Many thanks to the Reviewer for reviewing our manuscript.

This is an interesting manuscript but not entirely original. There are several methodological and experimental design issues that should be discussed in more depth.

I would like to thank the reviewer for his opinion, however, I believe that the work is original and even innovative because there are no similar works in the world literature relating to protein and especially amino acid changes in the course of naturally occurring mycotoxicosis on the background of Fusarium mycotoxins (deoxynivalenol and zearalenone) poisoning. Most of the available publications deal with other mycotoxins and, in addition, mycotoxicosis is induced artificially by deliberate administration of mycotoxins to cows. In our study, mycotoxicosis was induced naturally and all known amino acids were evaluated, which is a novel approach.

SPECIFIC COMMENTS

Lines 27-28: I don't see the results in the summary that allow us to conclude this. Also, talking about the "overall health of cows" is a bit risky and was not part of the study, or at least it was not sufficient and appropriately analyzed.

One sentence was changed, added “In cows with mycotoxicosis (baseline) showed low total protein, albumin, total-essential (TEAA) and total-non-essental amino acids (TNEAA) compared to later studies (p<0.01)”  Improvement in the overall health of the cows was shown which was added in the results. The overall health of the cows was evaluated, a description of which was added in the results. Therefore, the use of such a statement is justified in the revised form of the manuscript.

The explanation of the experimental design is somewhat confusing. As far as I understand, the authors used two dairy farms, one with mycotoxins in the diet was treated with the "deactivator" and another without mycotoxins was left as a control. If I properly understood the experimental design, I have some questions: a) was the presence of mycotoxins periodically evaluated in the diet of both farms? Were the cows included in the study balanced according to the number of lactations, daily milk production, body condition or other possible confounders? If this was not done, were these potential confounders included in the subsequent statistical analysis?

Some of this information was included in the L116-119 of manuscript. In addition, information on this has been added in the Results section.

The farms were different in terms of daily milk production and milk fat composition, how do the authors think these differences did not affect the treatment results? What was the daily milk production per cow?

The farms differed in the parameters mentioned by the reviewer because the herd in which cows were poisoned with mycotoxins was compared to a healthy herd. The aim of the study was to demonstrate the adverse effect of mycotoxins on selected parameters of protein metabolism and the general health of cows, which was obtained by comparing cows with mycotoxicosis (Exp. group) with healthy cows (Con. group). The aim of the study was also to check how the applied Micofix affected the evaluated parameters in cows in which mycotoxicosis was confirmed, which was demonstrated on the 30th and 90th day from the start of administration of this preparation. Also, comparing sick cows with healthy cows seems to be logical. There were no significant differences in the composition of fat, in the group Exp. 4.27 - 4.83% and the group Con. 4.51 - 4.84%. I am thankful to the reviewer for pointing out the fat content because in Con. group it was mistakenly underestimated, which has been corrected.

One of the farms presented significant health problems during the trial (judging from the data presented). Could this condition affect the results?

It may affect the results, it was the mycotoxins confirmed in these cows that caused the disease symptoms.

Table 2: Assuming the two groups were balanced (which they do not appear to be), the control group was not sampled at the same times as the treated group? You only have one data at the beginning of the trial, so how did the authors ensure that the variations found in the blood parameters of the treated cows are not physiological changes?

The material for testing (blood) from the cows of the control group was collected only once, as these were healthy cows in good condition with no changes in general health assessed by clinical examination, observation and interview data from the cow owners. The authors did not expect any changes in the parameters evaluated in the cows in the control group, since the cows were constantly in the same environmental conditions, in their herd and were fed the same diet throughout the study period, and did not receive the micotoxin deactivator, so it was unlikely that there would be any changes in subsequent tests beyond non-significant physiological changes and differences created in testing devices. Therefore, it was decided to collect a single sample in order to minimize the number of procedures performed in cows from the control group, which may adversely affect their welfare. Of course, we could have performed the tests in the control group three times, but I believe that it is not a mistake to perform the tests once so that there is a reference to the control group. It would be possible to present the studies described only as changes over time in the experimental group showing differences between individual samples, because there are clear differences. However, a comparison to a control group is required in all studies, so such a group was also included. It was different in experimental cows, in which mycotoxins were found in the serum and confirmed in the feed, these cows were also given a mycotoxin deactivator and in this group the authors expected changes, therefore the tests were performed 3 times.

Line 243: The evaluation of the health status of the cows was not part of the study, no results are presented, nor were statistical analyzes performed. In fact, the farm that had these symptoms at the beginning of the trial was the Control

The general health of the cows was assessed by clinical examination and observation of the cows.

In the Material and Methods, Animals section, the sentence "The assessment of the general health of all cows selected for the study was carried out on the basis of a clinical examination, clinical observations and data from the interview with the owners of the cows."

 

In the Results section, the results of clinical observations in cows in both groups have also been added.

Results of clinical examination and observation of cows with mycotoxicosis.

The observations were carried out for 3 months from the moment the feed contamination with mycotoxins was detected. Throughout this period, the cows were fed with the mycotoxin adsorbent - Micofix as an addition to the feed. In the first stage of the research, before starting the administration of the adsorbent, in experimental cows, many non-specific symptoms, which were already mentioned, were observed: decreased appetite, diarrhea, weight loss (BCS-3.0), lameness, reduced milk production (20.2±0.5 kg/head/day), increased number of somatic cells in milk (743±50.1x10³/mL), ovarian cysts were also occurring. After one month of using Micofix were shown: improved appetite, occasional diarrhea, improvement in cows mobility, slight increase in body weight (BCS-3.2), slight increase in milk production (21.0±0.5 kg/head/day) compared to the first study (p<0.003) and reduced somatic cell count in milk (536±30.2x10³/ml) compared to the first study (p<0.001). After 3 months of administration of Micofix, most of the symptoms subsided: the appetite returned to normal, the cows ate willingly, no diarrhea, increased body weight (BCS-3.5-4.0), no lameness, increased milk production (23.4 ± 1.0 kg/head/day) compared to the first and second studies (p<0.001), decrease in the number of somatic cells in milk (354 ± 20.3x10³/mL) compared to the first and second studies (p<0.001). Control cows were healthy BSC-4, milk yield (28.1 ± 1.0 kg/head/day) higher compared to all study dates in the experimental group (p<0.001), and somatic cell count in milk (325 ± 10.2x10³ / mL) lower compared to all study dates in the experimental group (p<0.001).

(accordingly, with earlier studies accepted for publication [30]).

Line 252: "Undoubtedly." With the experimental design used, the authors would not seem to be in a position to conclude with such emphasis, since two different farms were used and it was not (apparently) corrected for possible confounding factors.

Two farms were used because a control group was required. The control cows should be healthy, so it was decided to select this group of cows from another farm, but very similar to the one from which the experimental cows came. It would not be possible to create a control group in a herd that was fed feed contaminated with mycotoxins. I don't see any contradictions or confounding factors here.

The word ,,Undoubtedly” was changed to ,,Presumably”

Author Response File: Author Response.doc

Reviewer 2 Report

In this work, Marczuk et al. tried to find out the effects of supplementing a mycotoxin deactivator on the changes in protein metabolism indicators in dairy cows in response to mycotoxicosis. In the experimental design, the authors only collected the blood samples from experimental group at day 30 and day 90 after MDP, but no samples from control group. It was difficult to evaluate the change between particular dates of examinations were resulted from MDP effect or from time effect. Also, it is hard to understand why the protein metabolism indicators were important and chosen for evaluation in cows when in response to mycotoxicosis.  

Author Response

In this work, Marczuk et al. tried to find out the effects of supplementing a mycotoxin deactivator on the changes in protein metabolism indicators in dairy cows in response to mycotoxicosis. In the experimental design, the authors only collected the blood samples from experimental group at day 30 and day 90 after MDP, but no samples from control group. It was difficult to evaluate the change between particular dates of examinations were resulted from MDP effect or from time effect. Also, it is hard to understand why the protein metabolism indicators were important and chosen for evaluation in cows when in response to mycotoxicosis.

 

Thanks to the reviewer for the comment.

 

In the conducted studies, the effect of MDP was assessed on the basis of differences between the individual test dates in the experimental group, i.e. in cows with mycotoxicosis in which MDP was used. Samples for testing were taken after mycotoxicosis was detected and after 30 and 90 days of using Mikofix, the differences in some parameters were significant and clearly visible.

 

The material for testing (blood) from the cows of the control group was collected only once, as these were healthy cows in good condition with no changes in general health assessed by clinical examination, observation and interview data from the cow owners. The authors did not expect any changes in the parameters evaluated in the cows in the control group, since the cows were constantly in the same environmental conditions, in their herd and were fed the same diet throughout the study period, and did not receive the micotoxin deactivator, so it was unlikely that there would be any changes in subsequent tests beyond non-significant physiological changes and differences created in testing devices. Therefore, it was decided to collect a single sample in order to minimize the number of procedures performed in cows from the control group, which may adversely affect their welfare. Of course, we could have performed the tests in the control group three times, but I believe that it is not a mistake to perform the tests once so that there is a reference to the control group. It would be possible to present the studies described only as changes over time in the experimental group showing differences between individual samples, because there are clear differences. However, a comparison to a control group is required in all studies, so such a group was also included. It was different in experimental cows, in which mycotoxins were found in the serum and confirmed in the feed, these cows were also given a mycotoxin deactivator and in this group the authors expected changes, therefore the tests were performed 3 times.

 

Proper protein transformations in dairy cows are the basic metabolic mechanisms that determine both the maintenance of cow health and milk yield at the correct high level. The authors noticed a decrease in milk yield and some parameters of protein metabolism, e.g. total protein and albumin in cows with mycotoxicosis, which was interesting, which is why the authors decided to extend the research in these cows also to assess the level of amino acids in the blood serum. This was the basis for addressing protein and especially amino acid metabolism because there are no such studies in cows with naturally occurring mycotoxicosis.

Author Response File: Author Response.doc

Reviewer 3 Report

Why the control group was not sampled also at the 30^th and 90^th day after start of experiment? Results of these control cows (fed without “mycofix plus”)  would give clearer look on the development of researched parameters.

Why was not detected the concentration of mycotoxins in the serum of control cows?

Why was not detected the concentration of mycotoxins in the serum of experimental cows throughout experimental period, or at least at 30^th and 90^th day of experiment?

For better orientation in the manuscript, the names of sampling days must be alike. You used these different names for one sampling time: base line, initial and Before MDP administration.

Line 244 to 248: Based on what results can you assess this: … administration of Mycofix was associated with a gradual improvement in the health … ? Was it subjective evaluation?

Line 249: What you men with “first study”? If “before MDP administration”, than concentration of total protein and albumin/globulin ratio was not significantly lower. You cannot write in the text of manuscript “ was significant lower” whereas Table 1. shows no significant differences between control group and “Before MDP administration”.

Similar comment: Line 262 to 264: Sentence: After using Mycofix, there was a gradual increase in most of the described indicators (total protein, albumins, globulins and urea), which were comparable to the control group after a month. My Comment: Table 1. shows that concentration of total protein and globulins was between control group and 30^th day of experiment significantly different. Therefore you cannot write that concentration of total protein and and globulin was comparable to the control group after a month.

In both the results and the discussion, check that the description of significant differences between groups as well as between sampling times is interpreted correctly and in agreement with the data in the tables.

Improved function of liver is hypothesized. Why did you not determine also hepatic profile indices?

Brackets [] instead () why citing references in the text of manuscript.

Line 65 – remove “.” after word feed

 Conclusions: Can be evaluated health status of cows only based on results of this article? Please rewrite the conclusions only to results of this study.

Author Response

Many thanks to the Reviewer for reviewing our manuscript.

Why the control group was not sampled also at the 30^th and 90^th day after start of experiment? Results of these control cows (fed without “mycofix plus”)  would give clearer look on the development of researched parameters.

The material for testing (blood) from the cows of the control group was collected only once, as these were healthy cows in good condition with no changes in general health assessed by clinical examination, observation and interview data from the cow owners. The authors did not expect any changes in the parameters evaluated in the cows in the control group, since the cows were constantly in the same environmental conditions, in their herd and were fed the same diet throughout the study period, and did not receive the mycotoxin deactivator, so it was unlikely that there would be any changes in subsequent tests beyond non-significant physiological changes and differences created in testing devices. Therefore, it was decided to collect a single sample in order to minimize the number of procedures performed in cows from the control group, which may adversely affect their welfare. Of course, we could have performed the tests in the control group three times, but I believe that it is not a mistake to perform the tests once so that there is a reference to the control group. It would be possible to present the studies described only as changes over time in the experimental group showing differences between individual samples, because there are clear differences. However, a comparison to a control group is required in all studies, so such a group was also included. It was different in experimental cows, in which mycotoxins were found in the serum and confirmed in the feed, these cows were also given a mycotoxin deactivator and in this group the authors expected changes, therefore the tests were performed 3 times.

Why was not detected the concentration of mycotoxins in the serum of control cows?

The concentration of mycotoxins in the serum of cows from the control group was not determined due to the fact that mycotoxins were not detected in the feed consumed by these cows. For this reason, according to the authors, performing this assay was not necessary.

Why was not detected the concentration of mycotoxins in the serum of experimental cows throughout experimental period, or at least at 30^th and 90^th day of experiment?

In the experimental group, mycotoxin levels were determined to confirm mycotoxicosis after non-specific symptoms were found in the herd of cows and the presence of mycotoxins in the feed was detected. The authors only wanted to demonstrate the presence of mycotoxicosis in cows selected for the research group. However, after starting the administration of Mycofix, the authors decided that there is no need to test the level of mycotoxins, although the reviewer's remark is relevant and determining the level of mycotoxins on the 30th and 90th day could confirm the effectiveness of the deactivating effect of MDP. However, this was not done.

For better orientation in the manuscript, the names of sampling days must be alike. You used these different names for one sampling time: base line, initial and Before MDP administration.
The terms in the manuscript have been standardized and changed to ,,before MDP administration”.

Line 244 to 248: Based on what results can you assess this: … administration of Mycofix was associated with a gradual improvement in the health … ? Was it subjective evaluation?

The general health of the cows was assessed by clinical examination and observation of the cows.

In the Material and Methods, Animals section, the sentence "The assessment of the general health of all cows selected for the study was carried out on the basis of a clinical examination, clinical observations and data from the interview with the owners of the cows."

 

In the Results section, the results of clinical observations in cows in both groups have also been added.

 

Results of clinical examination and observation of cows with mycotoxicosis.

The observations were carried out for 3 months from the moment the feed contamination with mycotoxins was detected. Throughout this period, the cows were fed with the mycotoxin adsorbent - Micofix as an addition to the feed. In the first stage of the research, before starting the administration of the adsorbent, in experimental cows, many non-specific symptoms, which were already mentioned, were observed: decreased appetite, diarrhea, weight loss (BCS-3.0), lameness, reduced milk production (20.2±0.5 kg/head/day), increased number of somatic cells in milk (743±50.1x10³/mL), ovarian cysts were also occurring. After one month of using Micofix were shown: improved appetite, occasional diarrhea, improvement in cows mobility, slight increase in body weight (BCS-3.2), slight increase in milk production (21.0±0.5 kg/head/day) compared to the first study (p<0.003) and reduced somatic cell count in milk (536±30.2x10³/ml) compared to the first study (p<0.001). After 3 months of administration of Micofix, most of the symptoms subsided: the appetite returned to normal, the cows ate willingly, no diarrhea, increased body weight (BCS-3.5-4.0), no lameness, increased milk production (23.4 ± 1.0 kg/head/day) compared to the first and second studies (p<0.001), decrease in the number of somatic cells in milk (354 ± 20.3x10³/mL) compared to the first and second studies (p<0.001). Control cows were healthy BSC-4, milk yield (28.1 ± 1.0 kg/head/day) higher compared to all study dates in the experimental group (p<0.001), and somatic cell count in milk (325 ± 10.2x10³ / mL) lower compared to all study dates in the experimental group (p<0.001). (accordingly, with earlier studies accepted for publication [30]).

Line 249: What you men with “first study”? If “before MDP administration”, than concentration of total protein and albumin/globulin ratio was not significantly lower. You cannot write in the text of manuscript “ was significant lower” whereas Table 1. shows no significant differences between control group and “Before MDP administration”.

The sentence has been corrected to:

,,Protein metabolism indicators evaluated in the first study in cows with mycotoxicosis showed that the concentration of total protein, albumin, albumin/Globulin ratio, urea and total-non-essential amino acids (TNEAA) in experimental cows was lower (significantly albumin, urea and TNEAA) compared to control cows.”

The authors meant that the parameters listed in the first study in the experimental cows, i.e. before MDP administration, were lowered compared to the control group, but the reviewer correctly noted that not all parameters were statistically significantly lowered, so this sentence was corrected.

Similar comment: Line 262 to 264: Sentence: After using Mycofix, there was a gradual increase in most of the described indicators (total protein, albumins, globulins and urea), which were comparable to the control group after a month. My Comment: Table 1. shows that concentration of total protein and globulins was between control group and 30^th day of experiment significantly different. Therefore you cannot write that concentration of total protein and and globulin was comparable to the control group after a month.

The sentence was changed as suggested by the reviewer to: ,,After using Mycofix, there was a gradual increase in most of the described indicators (albumins, globulins, albumin/globulin ratio and urea), which were comparable to the control group after three months.”

In both the results and the discussion, check that the description of significant differences between groups as well as between sampling times is interpreted correctly and in agreement with the data in the tables.

As recommended by the reviewer, the significance of differences between groups and between sampling times were checked.

Improved function of liver is hypothesized. Why did you not determine also hepatic profile indices?

The hypothesis of improved liver function was related to the authors' own observations of decreasing levels of glycine, which is involved in the formation of bile salts and improved liver function, and the literature data cited by the authors about this [8,9]. In addition, the effect of mycofix declared by the manufacturer and the content of substances that improve liver function also allows for such assumptions. The authors focused on protein metabolism and clinical symptoms in cows with mycotoxicosis and did not include other results obtained during the study. Due to the complexity of metabolic processes occurring in dairy cows, especially during disease states, it is impossible to perform all the studies explaining the causes of changes in the results obtained, which is why the authors use literature data.

Brackets [] instead () why citing references in the text of manuscript.
corrected

Line 65 – remove “.” after word feed
corrected

Conclusions: Can be evaluated health status of cows only based on results of this article? Please rewrite the conclusions only to results of this study.

After making changes to the manuscript recommended by the reviewer, the conclusion is consistent with the research results. Additionally, the sentence that the reviewer pointed out was changed: ,,The study of protein metabolism parameters, especially the level of free amino acids, can not only be one of the factors used to assess the health status of cows in the herd, but can also be very helpful to properly balance the ration and possible supplementation with selected protected amino acids in cows with mycotoxin intoxication, which could ultimately significantly accelerate their recovery.”

Author Response File: Author Response.doc

Round 2

Reviewer 1 Report

The experimental design is confusing, and the explanations given by the authors failed to answer my questions. A herd was used as a control sampled only once, and it was "assumed" that the values would not change throughout the study because their general condition did not change. This may be so, but in an experimental trial, it is convenient to sample the control and experimental animals simultaneously.

The only control was the first sampling carried out on the animals of the experimental group (diet with mycotoxins), and they compared the evolution of the parameters by comparing them with the initial data. It is assumed that the observed changes are due to the use of the evaluated product. The analysis of animal health remains vague, there is not a single statistical reference on this point, and the data is presented subjectively.

I still believe there may be a positive effect due to the addition of the evaluated product, but this experimental design does not allow authors to conclude in this regard.

Author Response

Many thanks to the Reviewer for reviewing our manuscript.

The experimental design is confusing, and the explanations given by the authors failed to answer my questions. A herd was used as a control sampled only once, and it was "assumed" that the values would not change throughout the study because their general condition did not change. This may be so, but in an experimental trial, it is convenient to sample the control and experimental animals simultaneously.

I agree with the reviewer.

However, the material from the control cows was collected once, due to the welfare of the cows (in order not to unnecessarily expose cows to stress, which is very important in EU countries at the moment), these were healthy animals, they did not receive any preparations, therefore it was assumed that a single collection of the material would be sufficient.

The only control was the first sampling carried out on the animals of the experimental group (diet with mycotoxins), and they compared the evolution of the parameters by comparing them with the initial data. It is assumed that the observed changes are due to the use of the evaluated product. The analysis of animal health remains vague, there is not a single statistical reference on this point, and the data is presented subjectively.

I still believe there may be a positive effect due to the addition of the evaluated product, but this experimental design does not allow authors to conclude in this regard.

I also agree with the reviewer, the first sampling in the experimental group may be a reference to subsequent collections of material in this group.

The animal health assessment was based on clinical examination and observation of animals, therefore this assessment can be considered subjective, but it was always carried out by one veterinarian with 24 years of experience in working with dairy cows, i.e. me. The clinical trial includes an assessment of the health of cows based on CTO (heat, heart rate, respirations) and tests of individual body systems of the animal (respiratory, digestive, circulatory, nervous, reproductive, etc.), but the articles do not mention these tests, it is only written that the assessment of the state of health is carried out on the basis of a clinical examination and clinical observations. The observed changes in cow health, such as decreased appetite/improved appetite/normal appetite; diarrhea/occasional diarrhea/no diarrhea; lameness/improvement in cows mobility/no lameness; body weight loss/increase in body weight, are changes visible to the naked eye, and are also uncountable, therefore statistical evaluation does not seem necessary. The other tests in which numerical values were obtained (milk production, somatic cell count in milk) were subjected to statistical analysis, the same as for the other values, that is, for comparison with the control group, the Bonfferoni posthoc multiple comparison test was used and for comparison of values obtained in the experimental group at different test dates, Tukey's and Duncan's post-hoc tests were used, this is described in the paper in the statistical analysis section. In the results section, the obtained values and statistical significance are given.

I still believe there may be a positive effect due to the addition of the evaluated product, but this experimental design does not allow authors to conclude in this regard.

The conclusion was revised as recommended by the reviewer.

The conclusion has been changed to:

 

The presented results of own research in dairy cows with mycotoxicosis showed many non-specific symptoms: decreased appetite, diarrhea, weight loss, lameness, increased number of somatic cells in milk and ovarian cysts. In addition, in these cows, it showed low total protein, albumin, total-essential (TEAA) and total-non-essental amino acids (TNEAA). After 3 months of using the mycotoxin adsorbent, the disease symptoms subsided and the mentioned parameters of protein metabolism increased significantly. Based on the presented results, we can conclude that Mycofix is an effective preparation that inhibits the action of mycotoxins in the described scope of our research. From own research it can also be concluded that the study of protein metabolism parameters, especially the level of free amino acids, can not only be one of the factors used to assess the health status of cows in the herd, but can also be very helpful to properly balance the ration and possible supplementation with selected protected amino acids in cows with mycotoxin intoxication, which could ultimately significantly accelerate their recovery.

Author Response File: Author Response.doc

Reviewer 2 Report

I will still keep my previous comment and concern regarding the experimental design. I will leave the desicion to the editor.

Author Response

Thanks to the reviewer for the comment.

 

I will still keep my previous comment and concern regarding the experimental design. I will leave the desicion to the editor.

 

The previous reviewer's comment is answered in the previous responses to the review. I believe these responses are comprehensive.

Regarding the reviewer's current comment.

The authors have no concerns about the experimental design. The study was conducted properly in accordance with the applicable rules. According to the authors, these studies are very valuable because they combine basic research with clinical research and concern very rarely described changes in protein parameters - especially amino acids, during naturally occurring mycotoxicosis (DON and ZEA). For this reason, I believe that this work deserves to be published in a good journal like Agriculture.

Author Response File: Author Response.doc

Reviewer 3 Report

For authors

Thank you for correction of some of my comments.

I know, this is not my experiment. But, if the effect of mycotoxin deactivator is researched, the decrease of mycotoxins in blood of experimental animal is hypothesized. Therefore, the concentration of mycotoxin in the blood of experimental animals must be analysed (irrespective of animals welfare/wellbeing). Only this results can direct determined the effect of mycotoxins deactivator. Other analyses are indirect.

Similar as in my first review report, in the article are still two titles for one group: “first study” and “before MDP administration”. Pleas replace “first study” with “before MDP administration” in whole article.

Also similar as in my firs review report, in the article are disagreements between text and tables in significant differences. For example:

Sentence line 223-225: “The total protein concentration on the 30th and 90th day of the experiment was significantly higher compared to the baseline values before MDP administration (p ≤ 0,01).” Maybe you meaned “The total protein concentration on the 30^th and 90^th day of the experiment was significantly higher compared to the control group (p ≤ 0,01).“ I see (according to the footnotes under Table 1.) only these significance for total protein concentration.

Line 227: concentration of albumin on the 90^th day of the experiment was  similar with the control group. I think, similar will be more suitable word.

Line 317-319: This sentence is for me still confusing: “After using Mycofix, there was a gradual increase in most of the described indicators (albumins, globulins, albumin/globulin ratio and urea), which were comparable to the control group after three months.” What statistical method did you use to determine the gradual increase? In Table 1. I see no demonstrable differences between "Before MDP administration" "30th day of MDP use" and "90th day of MDP use" for parameters globulin, albumin/globulin ratio and urea. The only demonstrable increase is for albumin between "Before MDP administration" and "90th day of MDP use". Please be precise when describing the results. Do not confuse subjective opinion with statistical significance.

Author Response

Many thanks to the Reviewer for reviewing our manuscript.

For authors

Thank you for correction of some of my comments.

I know, this is not my experiment. But, if the effect of mycotoxin deactivator is researched, the decrease of mycotoxins in blood of experimental animal is hypothesized. Therefore, the concentration of mycotoxin in the blood of experimental animals must be analysed (irrespective of animals welfare/wellbeing). Only this results can direct determined the effect of mycotoxins deactivator. Other analyses are indirect.

I agree with the reviewer, it would have been good to evaluate the concentration of mycotoxins throughout the whole experiment in the experimental cows, but this was not assessed. The evaluation of the levels of the mycotoxins DON and ZEA was mainly carried out to confirm mycotoxicosis in cows in the experimental group, in order to classify the cows into the group with mycotoxicosis. The focus was not on the evaluation of mycotoxins, as the main purpose of the study was to evaluate protein metabolism in cows with mycotoxicosis before and after administration of the mycotoxin deactivator and to compare the values obtained with healthy cows.

 

Similar as in my first review report, in the article are still two titles for one group: “first study” and “before MDP administration”. Pleas replace “first study” with “before MDP administration” in whole article.

 

Once again, this was checked and corrected throughout the manuscript.

I'm sorry I overlooked that I didn't do it properly

Also similar as in my firs review report, in the article are disagreements between text and tables in significant differences. For example:

The manuscript was corrected according to the reviewer's suggestions to ensure that there are no inconsistencies in the text and data presented in the tables.

Sentence line 223-225: “The total protein concentration on the 30th and 90th day of the experiment was significantly higher compared to the baseline values before MDP administration (p ≤ 0,01).” Maybe you meaned “The total protein concentration on the 30^th and 90^th day of the experiment was significantly higher compared to the control group (p ≤ 0,01).“ I see (according to the footnotes under Table 1.) only these significance for total protein concentration.

It was changed to: ,,The total protein concentration on the 30th and 90th day of the experiment was significantly higher compared to the values before MDP administration and to the control group (p ≤ 0.01).”

Line 227: concentration of albumin on the 90^th day of the experiment was  similar with the control group. I think, similar will be more suitable word.

It was changed to ,,The level of albumin on the 90th day of the experiment was higher compared to the values before MDP administration (p ≤ 0.01), and similar to the control group. The lowest albumin concentration was obtained from the samples before MDP administration.”

Line 317-319: This sentence is for me still confusing: “After using Mycofix, there was a gradual increase in most of the described indicators (albumins, globulins, albumin/globulin ratio and urea), which were comparable to the control group after three months.” What statistical method did you use to determine the gradual increase? In Table 1. I see no demonstrable differences between "Before MDP administration" "30th day of MDP use" and "90th day of MDP use" for parameters globulin, albumin/globulin ratio and urea. The only demonstrable increase is for albumin between "Before MDP administration" and "90th day of MDP use". Please be precise when describing the results. Do not confuse subjective opinion with statistical significance.

It was changed to: ,, After using Mycofix for three months, there was an insignificant increase in most of the described indicators, and only in the case of albumin the increase was statistically significant, which were comparable to the control group.”

Author Response File: Author Response.doc